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the performance of ambulatory surgical procedures is on the rise across all surgical fields , ranging from thyroid surgery1 to cholecystectomy.2 the field of orthopedic surgery has followed a similar path , with an ever - increasing practice trend of outpatient knee and shoulder arthroscopy3 4 and more recently lumbar and cervical spine surgery.5 6 7 8 given these trends , we sought to assess the outpatient spine surgery environment and report the types of cases being performed by those surgeons who perform spine surgery in this setting . we conducted a survey of spine surgeon members of the international society for the advancement of spine surgery ( isass ) regarding their experience with ambulatory spine surgery . in so doing , we hoped to characterize the current practice of spine surgeon members of this society , including the characteristics of respondents performing ambulatory surgery , the surgical procedures being performed , the setting of ambulatory surgery as well as the associated self - reported complications encountered during the performance of ambulatory surgery . the electronic survey consisted of 25 questions and was distributed to members of the isass over a 3-month period from july through september 2012 . after this time , the response web link was disabled . by providing each respondent with a unique link , we avoided multiple responses from a single participant . for the analysis of factors potentially associated with ambulatory spine surgery , data were first examined in a univariate manner using the student t test for continuous variables and fisher exact test for discrete data . for the multivariate analysis , variables with a p value < 0.25 were entered into a multiple logistic regression model , because we interpreted these variables as independent factors associated with the event or outcome of interest , over and above ( adjusted for ) other potential factors included in the equation . the logistic equation generates p values and odds ratios for each explanatory variable 's association with the outcome of interest . for all statistical analysis , data were analyzed using the sas system software version 9.2 ( sas institute , inc . , the p values were not adjusted for multiple testing and a potential inflation of the type i error . we found that 75.4% of respondents were trained in orthopedic surgery , with the remainder having been trained in neurosurgery . in addition , 87.7% of surgeon respondents were spine fellowship trained ; 61.4% were in private practice , 31.6% in academic practice , and 7.0% in a hospital employment position . the majority ( 54.4% ) classify themselves as practicing in an urban environment , with 42.1% in a suburban environment and 3.5% in a rural area . 84.2% of respondents performed some manner of ambulatory spine surgery , whether in a hospital or ambulatory surgery center setting . of the responding surgeons , 49.1% invest in an ambulatory surgery center ; of those who perform surgery in such a center , 81.5% are investors ; and in those performing ambulatory surgery in a hospital setting only , 21.1% invest in a surgery center ( table 1 ) . common procedures were single- ( performed by 70.8% of surgeons ) or multiple - level ( 41.7% ) lumbar microdiskectomy , single- ( 62.5% ) or multiple - level ( 33.3% ) lumbar laminectomy , and one- ( 54.2% ) and two - level ( 39.6% ) anterior cervical diskectomy and fusion . surgeon investors in ambulatory surgery centers were more likely to perform procedures of increased complexity ( i.e. , multilevel anterior cervical fusion procedures ) than noninvestors ( 21.4% versus 3.4% ) ; in other words , of those performing such procedures , 85.7% were investors . the numbers in this analysis were too small to perform statistical analysis . surgeons in private practice were more likely to perform ambulatory surgery ( 94.3% ; p = 0.0176 ) , and nonacademic surgeons ( i.e. , those in private practice or community hospital - based ) were more likely to invest in ambulatory surgery centers ( 67.6% ; p = 0.0024 ) and perform surgery at least part of the time in a surgery center ( p = 0.0039 ; table 2 ) . in the univariate analysis , status as an orthopedic surgeon ( versus a neurosurgeon ) did not correlate with performance of outpatient surgery ( p = 0.5084 ) , with investment in an ambulatory surgery center ( p = 0.3084 ) , or with the location of the ambulatory surgery ( p = 0.9798 ; table 3 ) . of note , taken together as a group , being in practice for 20 years or less did correlate with the likelihood of investing in a surgery center ( p = 0.0333 ; table 2 ) . location of performance of ambulatory surgery did not appear to affect whether the primary surgeon co - operated with another surgeon . among those performing ambulatory surgery at least sometimes in ambulatory surgery centers , 48.3% reported the availability of 23-hour observation should the patient require it ; the remainder indicated that transfer to another facility would be necessary for further care . in addition , 10.3% of surgeons reported a complication that could not be addressed in the ambulatory center environment ; 92% noted that in the event of such a complication , there was a protocol in place designed to manage such episodes . p < 0.05 ( statistically significant ) . based on measurement of suburban rather than urban location cervical and lumbar spine surgery is being performed more commonly on an ambulatory basis,5 6 7 8 possibly driven by the development of minimally invasive techniques , which has been shown to minimize immediate postoperative pain and accelerate postoperative recovery.9 10 11 another factor may be surgeon financial incentive when performing the surgery in a physician - owned ambulatory surgery center.12 this survey was able to provide an overview of the characteristics of surgeons performing surgery on an outpatient basis , the location for the performance of the surgeries , and the types of cases being performed . based on the data gathered , practicing as a member of a nonacademic practice correlated with the performance of ambulatory surgery , the utilization of an ambulatory surgery center , and investment in an ambulatory surgery center . likewise , being in practice for 20 or fewer years and being a member of a nonacademic practice correlated with investment in an ambulatory surgery center . finally , as noted above , though the numbers were too few to calculate statistical significance , there was a trend toward performance of both surgery in ambulatory surgery centers and procedures associated with increased risk ( i.e. , multilevel anterior cervical fusion procedures ) on an ambulatory basis being undertaken largely by investors in an ambulatory center . given the financial incentives involved in an ambulatory surgery center , it is possible that this plays a role in the decision to perform these procedures in this setting versus that of a hospital , where a patient may have better access to care should a postoperative complication arise requiring emergent assessment and treatment by a physician . of some concern is that 8% of surgeons performing spinal procedures did not have a mechanism for dealing with complications that could not be managed in the ambulatory surgery center . last , 10.3% of surgeons identified complications that could not be handled in their center . limitations to our study include those inherent to survey studies ( sampling error , nonresponse error , coverage error ) and a relatively small number of respondents . ideally , a study such as ours would include survey distribution to a wider range of spine surgeon professional societies , providing a more thorough and accurate analysis of the patterns of performance of spine surgery in the ambulatory setting . the authors undertook this study not with the intention of criticizing the use of ambulatory surgery centers for spine surgery as a whole , but with the hope that this study will serve to open discussion on the types of procedures that can be safely performed in an ambulatory surgery center . this discussion should divest itself of financial incentives and focus entirely on patient safety and mechanisms to deal with complications that can not be managed in ambulatory centers .

habitual dislocation of the patella is a rare condition among adults , where the patella dislocates during flexion and relocates during extension without pain and swelling unlike the recurrent patellar dislocation . these techniques are all designed to relocate the patella and regain alignment of the extensor mechanism . determination of the factors permits an effective elective treatment plan and it has been known that satisfactory or significant treatment results can be obtained with combined procedures , not with a single procedure . we report a case of iatrogenic habitual dislocation of patella with osteoarthritis of left knee in a 50-year - old female patient , who was treated with lateral release quadricepsplasty , medial patello femoral ligament ( mpfl ) reconstruction and tibial tuberosity transfer . a 50-year - old woman presented with iatrogenic habitual dislocation of left patella from childhood and pain from past 3 years . she recalled a childhood surgery over middle third of left thigh ( incision and drainage ) . few years after the surgery , she started noticing the giving way of patella and lived for 38 years without much disability . she was carrying out her daily activities without much pain or symptoms , but she was never symptom free during this period . there was a healed surgical scar over middle and lower third lateral aspect of the left thigh . although the patella was in the femoral trochlear groove in extension , it dislocated laterally with 25 degrees of knee flexion . she complained slight discomfort by an apprehension test , but was not very painful even when dislocated . the passive lateral patellar tilt test was negative since the soft tissue attachment to the lateral border of the patella was diffusely stiff and tight . radiological examination revealed evidence of patella alta as defined by insall and salvati , and q angle was 18 degrees . clinically and radiologically ( grade iii kellgren and lawrence classification ) tricompartmental degenerative knee arthritis was appreciated [ figure 1 ] . preoperative radiographs anteroposterior ( a ) and lateral view ( b ) of left knee joint showing degenerative changes . ( c ) skyline view of normal knee ( right ) ; ( d ) skyline view of affected left knee showing lateral subluxation of patella with patient in supine position under epidural anesthesia , an anterior midline knee incision was used with paramedian arthrotomy . the lateral tether is released by dividing the fibrous adhesions , which extended along the lateral intermuscular septum . the lateral patellar retinaculum and the synovium were divided and vastus lateralis was detached from the quadriceps tendon , leaving a rim of tendon for suturing . due to shortening of the extensor mechanism , the patella could not be reduced . tracking was augmented with mpfl reconstruction using semi - tendinosus tendon and patellar end was fixed with 2.8 mm fastak titanium anchor on the medial border of patella and over near isometric point on medial epicondyle of femur , which was checked under fluoroscopy . patellar maltracking was persistent and so , it was decided to proceed with medial and distal transfer of tibial tuberosity . a long - leg hinged knee brace was applied with the knee in 20 of flexion and partial weight bearing with crutches for four weeks was advised , during which the patient was encouraged to do vigorous static quadriceps strengthening exercises . knee mobilization and full weight bearing was started after four weeks . but after vigorous quadriceps exercises , patient improved without any extensor lag . at final followup two years radiographs showed well reduced patella in both lateral , anteroposterior view and skyline view [ figure 2 ] . the patient 's kujala knee society score 89 [ figure 3 ] and range of motion was 0 to 90 on the left side [ figure 4 ] . postoperative radiographs anteroposterior ( a ) and lateral views ( b ) of left knee showing tibial tuberosity transfer fixed with screws and 2.8 mm fastak titanium anchor on the medial border of patella ( c ) skylinr view of left side showing fastak titanium anchor ( d ) skyline vfiew of normal knee ( right ) clinical photographs showing functional outcome ( a ) walking down the stairs ( b ) sitting cross legged clinical photographs showing range of motion ( a ) flexion of 90 degrees and ( b ) extension at left knee ( postoperatively ) with patient in supine position under epidural anesthesia , an anterior midline knee incision was used with paramedian arthrotomy . the lateral tether is released by dividing the fibrous adhesions , which extended along the lateral intermuscular septum . the lateral patellar retinaculum and the synovium were divided and vastus lateralis was detached from the quadriceps tendon , leaving a rim of tendon for suturing . due to shortening of the extensor mechanism , the patella could not be reduced . tracking was augmented with mpfl reconstruction using semi - tendinosus tendon and patellar end was fixed with 2.8 mm fastak titanium anchor on the medial border of patella and over near isometric point on medial epicondyle of femur , which was checked under fluoroscopy . so , it was decided to proceed with medial and distal transfer of tibial tuberosity . a long - leg hinged knee brace was applied with the knee in 20 of flexion and partial weight bearing with crutches for four weeks was advised , during which the patient was encouraged to do vigorous static quadriceps strengthening exercises . knee mobilization and full weight bearing but after vigorous quadriceps exercises , patient improved without any extensor lag . at final followup two years radiographs showed well reduced patella in both lateral , anteroposterior view and skyline view [ figure 2 ] . the patient 's kujala knee society score 89 [ figure 3 ] and range of motion was 0 to 90 on the left side [ figure 4 ] . postoperative radiographs anteroposterior ( a ) and lateral views ( b ) of left knee showing tibial tuberosity transfer fixed with screws and 2.8 mm fastak titanium anchor on the medial border of patella ( c ) skylinr view of left side showing fastak titanium anchor ( d ) skyline vfiew of normal knee ( right ) clinical photographs showing functional outcome ( a ) walking down the stairs ( b ) sitting cross legged clinical photographs showing range of motion ( a ) flexion of 90 degrees and predisposing factors include ligamentous laxity , contracture of the lateral patellar soft tissues , patella alta , quadriceps contractures , hypoplasia of the lateral femoral condyle and genu valgum ( bony factors ) . the soft tissue and bony structures mentioned above give an active and passive stability , which allow the patella to be located within the trochlea during knee movements . permanent patellar dislocation , either congenital or acquired , is diagnosed at an early age in childhood and numerous surgical techniques have been introduced for its treatment . this is not the same for adult patellar dislocation with arthritis , where no specific treatment protocols are established . however , it has been known that combined procedures , not one single procedure , should be performed to achieve relatively satisfying treatment results . in our case , the patient was a 50-year - old female with moderate degeneration and an active lifestyle . hence , tkr was an option and we had decided to go for staged procedures - initially getting the normal patellar tracking and later tkr . when patellar realignment is selected for habitual dislocation of the patella with quadriceps contracture in adults , the gold standard of management has not yet been established . successful results require combination of these procedures , depending on clinical and intraoperative findings on an individual basis . habitual dislocation is associated with shortening of the quadriceps muscles and considering that lengthening of the tendon is an essential part of the procedure to allow the patella to remain reduced after realignment . in our case , as a result of childhood surgery and long term dislocation , the patient had quadriceps contracture and lateral soft tissue contractures , hence we decided to proceed with lateral release and quadriceps plasty as initial procedure . medial vector augmentation and patellar tendon alignment procedures should be included depending on the degree of quadriceps dysfunction . many times in neglected dislocations , even after lateral release once the knee joint flexion starts , patella tends to dislocate laterally . to prevent this , , we have done medial patello - femoral ligament reconstruction using semi - tendinosus tendon . medial transfer of tibial tuberosity was decided intraoperatively after doing above mentioned three procedures , because the patellar tracking was not satisfactory although the q angle was near - normal . the diagnostic relevance of q angle as an indication for distal realignment was not established in our case and we recommend tibial tuberosity - trochlear groove distance ( tttg ) as measured by ct scan as a better tool . although standard procedures of patellar realignment include simple lateral release , proximal realignment , distal patellar tendon realignment or combination of them , addressing the pathoanatomy which is unique for every individual and careful pre - operative evaluation would give a better treatment algorithm . marmor was the first to perform tka in an adult patient with congenital dislocation of the patella without reconstructing the extensor mechanism . he reported replacement of the patella be avoided in adult patients with congenital dislocation of the patella if they can function reasonably well . reported tka for bilateral congenital dislocation of patella performed on the basis of marmor 's report . unconstrained tka with extensive soft tissue release in the right knee failed after 14 months . total knee arthroplasty in middle - aged patient with habitual dislocation is not a permanent solution . though the initial plan was to subject the patient for total knee replacement after soft tissue surgery , the procedure was deferred as there was significant improvement in function ( kujala score ) after the soft tissue surgery and the patient herself denied the second procedure . we believe that the soft tissue surgery as primary procedure will buy additional time for patient to delay the tka and proper soft tissue balancing will improve the longevity and clinical outcome of the definitive procedures .

carpal tunnel syndrome ( cts ) is the most common entrapment neuropathy caused by the compression of the median nerve in the carpal tunnel . common complaints are painful paresthesias or burning pain in the lateral half of the hand and the radial three fingers [ 2 , 3 ] . diagnosis is performed with the history , physical and neurological examination , electromyography , and nerve conduction velocity measurements . in the management of cts , conservative procedures and surgical approaches the aim of the surgery is to release the transverse carpal ligament and thereby to increase canal volume and reduce the pressure over the median nerve . nonendoscopic procedure includes a standard open technique ( conventional ) , a wrist - incision technique , and midpalmar - incision technique . in the present study , we aimed to compare the surgical results of two nonendoscopic different techniques , in the eastern part of turkey , retrospectively . this study included 200 patients who were diagnosed with cts in bitlis state hospital between may 2011 and may 2012 . 95 patients who did not benefit from conservative management and electromyography findings revealed moderate , moderate - severe , and severe were operated . folders of these 95 patients were retrospectively searched for incision type , need of reoperation , scar - related problems , any intraoperative complication , and the time of starting to use their hands in daily activities . operations were performed by two different neurosurgeons ( ig , ha ) according to their own experience . both types of incision were made under local anesthesia . first incision type ( group i , operator ha ) was shortened conventional type in which an incision beginning from distal wrist crease and extending distally about 2 to 2,5 cm over the carpal canal was made ( figure 1 ) . after that with blunt dissection transverse carpal ligament is found and cut until the distal end . second incision type ( group ii , operator ig ) was transverse mini - incision in which a transverse incision was made 1,52 cm in length about 1 cm above distal wrist crease ( figures 2 and 3 ) . after incision , palmaris longus tendon was identified laterally to the median nerve on the anterior surface of the wrist . after that the median nerve was released blindly . this retrospective study included 95 patients . 87 ( 91,5% ) of them were female and 8 ( 8,5% ) were male . 50 of them were been operated with shortened conventional incision , group i , and 45 of them were been operated by horizontal incision , group ii . mean age was 53,76 9,0 years in group i and 52,60 8,0 years in group ii . we detected that 4 ( 8% ) patients in the first group were rereferred with the scar tissue problem at the wound site and were treated with the topical application of mupirocin containing cream . in the second group , no problems were determined associated with the formation of scar tissue . when two groups were compared in terms of the formation of scar tissue at the wound site , there was no statistical difference ( p = 0,07 ) . we determined that in a patient in the second group transverse mini - incision technique had been converted to classical conventional technique due to uncontrolled digital artery bleeding . also , we detected ulnar artery injury in a patient in the second group . we noted that this patient complained of sensory loss in the same hand up to anesthesia about one month after operation . this patient was reoperated due to anesthesia and severe cts findings in emg with the classical conventional technique . additionally , we determined that a patient in the same group reoperated due to recurrence of preoperative complaints and severe cts findings in emf 3 months after operation with classical conventional method . as a result , the need of surgery with classical conventional technique was determined in total of three patients in the second group . however , no statistically significant difference was observed in terms of need of classical conventional technique ( p = 0,5 ) . also , there was no significant difference in terms of intraoperative complication rates ( p = 0,47 ) . the use of patients ' hands after operation in daily activities was 22.28 9.4 days in group i and 15,96 7,4 days in group ii . statistically significant difference was observed between two groups in terms of starting to use hands in daily activities ( p = 0,02 ) . parametric values were given as a mean standard deviation and nonparametric values were given as a percentage . to compare parametric continuous variables , student 's t - test was used ; to compare nonparametric continuous variables , the mann - whitney u test was used . two - tailed p values of less than 0.05 were considered to indicate statistical significance . since the first description of operative treatment of cts , which is the most common peripheral entrapment neuropathy , it has become a profession which neurology clinicians , neurosurgeons , orthopedic surgeons , and plastic surgeons deal with it . according to this fact , different but near to each other , ideas have been reported in the management of cts . in 1993 , the american academy of neurology 's official practice guidelines reported that in the management of cts , noninvasive options should be the first choice and surgery should be kept for refractory cases . the american academy of orthopedic surgeons recommended both of the noninvasive and surgical treatments for early cts without denervation of the median nerve . conservative options for cts include nonsteroidal anti - inflammatory drugs , local steroid injection , wrist splinting , activity modification , exercises , physical therapy modalities , and alternative therapies . in our clinic , routine practice is that symptomatic patients whose emg findings reveal mild and moderate cts findings are firstly treated with conservative approaches and we take support from physical therapy and rehabilitation department . however , patients whose emg findings reveal severe cts findings are advised to be operated . the surgical options for cts may be divided into two categories . these are endoscopic and nonendoscopic procedures . nonendoscopic procedures may be summarized as standard open technique ( conventional ) , a wrist - incision technique , and midpalmar - incision technique . endoscopic procedures for cts were firstly used in 1989 and aimed at the advantage of decreased postoperative pain and faster return of patients to their work [ 911 ] . however , in a randomized controlled study which is comparing endoscopic surgery with open surgery for cts , atroshi et al . reported that endoscopic surgery was associated with less postoperative pain . however , the small size of the benefit and similarity in other outcomes make its cost effectiveness uncertain . endoscopic approach is not risk free and incomplete relief or recurrence of symptoms due to incomplete decompression . also , injury to the superficial palmar arch or median or ulnar nerve was been reported in the literature [ 5 , 11 , 1321 ] . additionally , endoscopic procedures require costly equipment , technical support , and prolonged operative setup time . the classical conventional surgery for cts provides the surgeon full inspection of the transverse carpal ligament . also , contents of the carpal tunnel and the presence of any intraligamentous motor nerve branch to the thenar muscles may be seen easily with this procedure . the optimal incision length in this procedure depends on patient- and surgeon - related factors . according to traditional definition , this surgery is performed with a longitudinal incision extending from kaplan 's cardinal line distally to beyond the wrist crease proximally , about 4 - 5 cm in length . however , nowadays this approach is performed with an incision 24 cm in length , which ends approximately 2 cm distal to the wrist crease . conventional surgery has been the mainstay in the surgical management of cts for several decades and its success rates in terms of alleviation of patient symptoms range between 81 and 98% with minimal complications [ 2227 ] . wrist - incision technique is performed with a incision which is 1 cm above distal wrist crease and transverse carpal ligament is cut blindly from proximal to distal end . however , in midpalmar incision , ligament is cut distally to proximally [ 5 , 9 , 2830 ] . the aim in both of these procedures is to decrease pillar pain or scar tenderness . it was reported that outcomes were similar to those of the endoscopic approach [ 5 , 9 ] . in a study comparing limited palmar incision with conventional technique , authors concluded that limited palmar incision is as effective and safe as conventional technique . moreover , they concluded that limited palmar technique had better postoperative recovery and cosmetic results . in a prospective randomized study comparing limited open techniques with endoscopic procedure , authors found that results of both groups were similar to each other at the end of one year . however , patients complained of less tenderness of the scar at the second and fourth postoperative weeks in limited open release using the strickland instrumentation group . additionally , less thenar and hypothenar pain was reported in the same group . in our study , four patients in conventional group complained of scar tenderness ; however , there was no statistically significant difference . conventional method is being performed with an incision 24 cm in length nowadays and this contributes to better cosmetic results . in our patients , we also used an incision which was 2 - 3 cm in length according to patient - related factors . in our study , the most important difference between two groups was in the time of using their hands in daily activities . conventional technique always will be an effective and safe method for patients who will be operated for the first time or recurrence.in patients who especially care about cosmetic results and return to work , transverse mini - incision technique may be a safe alternative in experienced hands . conventional technique always will be an effective and safe method for patients who will be operated for the first time or recurrence . in patients who especially care about cosmetic results and return to work , transverse mini - incision technique may be a safe alternative in experienced hands .

as part of a larger study examining the immunopathogenesis of severe bronchiolitis , nonbronchoscopic bronchoalveolar lavage samples were collected from 30 infants ventilated with rsv bronchiolitis ( determined by antigen detection in nasopharyngeal aspirates ) in 2000 and 2001 . briefly , a suction catheter was passed down the endotracheal tube beyond the bifurcation of the bronchi . two 1-ml / kg aliquots of sterile normal saline were instilled separately down the suction catheter . control samples were obtained in winter from children within the same age range who were being intubated and ventilated for noninfection - related conditions . the local research ethics committee approved the study , and informed consent was obtained from parents . detection of the human metapneumovirus genome was performed by reverse transcription - polymerase chain amplification ( rt - pcr ) of the matrix ( m ) , fusion ( f ) , and nucleoprotein ( n ) genes . rna was extracted from the specimens by the guanidine / silica method ( 8) , and 15 l was added to a 50-l rt reaction with a polyt primer and 1.25 u amv - rt ( gibco , basingstoke , u.k . ) to produce cdna . each pcr reaction contained 10 l of cdna with 2.5 u amplitaq gold ( applied biosystems , warrington , u.k . ) , 2.0 mm mg cl2 , 300 m deoxynucleoside triphosphate , 0.4 m of each primer , and the buffer supplied . for amplification of the m gene , primers hmpv - mf1 ( aag tga atg cat cag ccc aag ) and hmpv - mr1 ( cac aga ctg tga gtt tgt caa a ) , which amplified the region between nucleotides 212 and 331 , were used to give an amplicon of 120 bp . pcr cycling conditions were 95c for 10 min followed by 45 cycles of 95c for 1 min , 58c for 1 min , 72c for 1 min , and a final extension step of 72c for 10 min in a perkin - elmer ( warrington , cheshire , u.k . ) positive samples were confirmed by pcr amplification for the f or n gene products , or by amplification of a longer product of the m gene . the longer m product used primer hmpv - mr1 and a new primer , hmpv - mf2 ( atg gag tcc tat cta gta gac a ) , amplifying the region between nucleotides 1 and 331 of the m gene , yielded a 331-bp product . for the f pcr , primers hmpv - ff1 ( gtg agc tgt tcc att ggc ag ) and hmpv - fr1 ( ccc tca act ttg ctt agc tga ta ) amplified the region between nucleotides 1162 and 1295 of the f protein to give a 134-bp pcr product . pcr cycling conditions were as for the m protein but with an annealing temperature of 62c . the n pcr used primers hmpv - nf1 ( gta tta cag aag ttt gtt cat tga g ) and hmpv - nr1 ( gag aac aac act tgc aaa gtt gg ) , which amplified the region between nucleotides 710 and 1034 of the n protein to give a 325-bp product . in each case , the primers were designed by aligning the m , n , and f gene sequences of the original dutch isolates ( 93.1 , 93.2 , 93.3 , 94.1 , 94.2 , 99.1 , 99.2 , and 00.1 ) ( 3 ) to define regions that were held in common . selected pcr products were cloned into a ta cloning vector ( pgem - t , promega , southampton , hampshire , u.k . ) , and the sequence was determined to confirm the identity of the virus detected by the pcr reaction . sequences from the m , n , and f proteins of one virus ( liv08 ) have been deposited in embl , with the accession numbers aj439505 , aj439504 , and aj439503 , respectively . the phylogenetic relationship between isolates human metapneumovirus sequences were aligned with the clustalx multiple alignment program ( embl , heidelberg , germany ) , and analyses were carried out by using the phylogeny inference package ( phylip version 3.5c , ( j. felenstein and the university of washington ) . dna sequences were analyzed by using dnadist followed by neighbor joining ; 100 replica samplings were analyzed . detection of rsv by rt - pcr and n gene typing was as described ( 9 ) . m gene and at least one other of n , f , or long m amplicons of human metapneumovirus were detected in bronchoscopic bronchoalveolar lavage fluids from 21 ( 70% ) of 30 infants ventilated for rsv bronchiolitis ( table ) . amplicons were cut from agarose gels , eluted , and purified by using a gel purification kit ( qiagen , west sussex , u.k . ) . the amplicons were cloned into pgem - t ( promega ) , and sequencing was conducted with mi3 primers by lark technologies ( essex , u.k . ) . in each case , the amplicons were very similar ( 1 base different ) to each other and closely related to those reported for human metapneumovirus ( 82% to 97% ) ( 3 ) . the figure shows a neighbor - joining tree that uses a 102-bp ( nucleotide 208309 ) region of the m gene , demonstrating the relationship of a liverpool ( liv08 ) strain to those from holland . rsv was detectable by rt - pcr in only 24 ( 80% ) of 30 nonbronchoscopic bronchoalveolar lavage samples , despite rsv antigen s being detectable from all nasopharyngeal aspirates at the onset of illness . of these 24 , 23 could be assigned an n genotype ( 18 np-4 , 4 np-2 , 1 np-3 ) . human metapneumovirus coinfection was detectable with each of the genotypes , although only one ( 25% ) of the four infants infected with np-2 was coinfected with human metapneumovirus , compared with 13 ( 76% ) of 17 of those with np-4 . human metapneumovirus was detectable in three infants from whom no rsv amplicon could be obtained . neither rsv nor human metapneumovirus amplicons were detectable in nonbronchoscopic bronchoalveolar lavage fluid from 10 control patients ventilated for reasons unrelated to bronchiolitis . most infants have serologic evidence of infection with rsv by 2 years of age ( 10 ) , but other than host factors such as underlying cardiac , respiratory , or genetic factors and prematurity , why severe disease develops in some infants and mild disease in others is unclear . for example , although 87% of children have antibodies to rsv by age 18 months ( 11 ) , most children have only upper respiratory tract symptoms such as rhinitis , cough , and coryza ( 12 ) . the age of the patient is also important because lower respiratory tract signs develop in up to 40% of infants < 1 year , and 0.5% to 2% of all infants need to be hospitalized ( 12 ) . coinfection with rsv and other pathogens has been described previously . in studies in northern ireland ( 13 ) and india ( 14 ) , coinfection occurred in 1.1% ( all with rhinovirus ) and 15% ( with influenza and parainfluenza viruses ) of patients with rsv acute lower respiratory tract infection , respectively . however , apart from in viral infections that might alter host immunity ( 15 ) , acute respiratory tract infections involving rsv alone were not significantly different from those caused by rsv and another virus ( 16 ) . rsv and human metapneumovirus might have similar seasonal patterns ( 3 ) , so coinfection is possible . in the present study , we detected coinfection with human metapneumovirus in 70% of infants with rsv bronchiolitis sufficiently severe to require admission to the pediatric intensive - care unit for ventilatory support . , patient ; rsv , respiratory syncytial virus ; hmpv , human metapneumovirus ; bpd : bronchopulmonary dysplasia ; bal , bronchoalveolar lavage ; np , nucleoprotein genotype the phylogenetic relationship of human metapneumovirus from liverpool ( liv08 ) to those from holland and to avian pneumovirus . sequence analysis of a portion of the m gene from four of the detected human metapneumoviruses indicated that they were closely related both to each other and to the dutch isolates . this high rate of coinfection raises the possibility that coinfection with rsv and human metapneumovirus might be another determinant of rsv disease severity . indeed , of 10 studied infants with severe rsv bronchiolitis and no other risk factors for severe disease , 9 ( 90% ) were coinfected with human metapneumovirus . confirmation of a role for coinfection will require longitudinal studies over several bronchiolitis seasons , comparing disease severity with frequency of coinfection across the full spectrum of rsv disease . finally , this high rsv human metapneumovirus coinfection rate in severe disease means that previous studies into rsv subtype / genotype , disease severity , and cytokine and chemokine expression in rsv disease may need to be reevaluated .

vascular calcifications have been associated with adverse clinical outcomes in ckd patients , including ischaemic cardiac events , claudication and mortality [ 16 ] . it does not only consist of a simple precipitation of calcium ( ca ) and phosphorus ( p ) , but it is also an active and modifiable process . vascular calcification in ckd may be interpreted as a result of the dysregulation of the equilibrium between calcification promoters and inhibitors in which several uraemic factors , including abnormalities in the mineral metabolism , are implicated [ 711 ] . other risk factors for vascular calcification , such as age , diabetes mellitus , dyslipidaemia , hypertension and smoking , may play a role not only in vascular calcification but also in bone health . bone mineral density and osteoporotic fractures are well - known markers of bone health in both general and ckd populations . however , until recently , they had not been linked or associated with changes in the vascular system . experimental data have clearly shown that vascular smooth muscle cells can modify their phenotype becoming osteoblast - like cells able to induce vascular calcifications [ 1419 ] . this fact has opened a fascinating area of cross - link research between vascular and mineral metabolisms . in addition , recent data from general populations and ckd patients have shown that not only vascular calcifications but also osteoporotic fractures are associated with an increased risk of mortality . the aim of this multicentre study carried out in haemodialysis ( hd ) patients was to investigate the association between vascular calcifications , osteoporotic fractures and survival in hd patients . the study was carried out in 193 hd patients ( 121 men and 72 women ) from a total of 258 patients from 7 hd units in asturias ( spain ) followed up for 2 years.the clinical practice protocols and the type of dialysis in all dialysis units were similar , and the quality control of aluminium ( al ) in dialysis water in all units was performed in the reference centre ( hospital universitario central de asturias ) . once the informed consent was obtained , only the hd patients who completed the radiological studies were subsequently included in the study . patients who did not give informed consent and patients with senile dementia were not included in the study . the mean age was 64 14 years ( men ) and 68 13 years ( women ) ; the mean time on hd was 3.1 3.6 years . most women ( 91.7% ) were post - menopausal but had never received oestrogen , selective oestrogen receptor modulators or bisphosphonates . vascular calcifications and vertebral fractures eighty - four different parameters , including clinical , biochemical and therapeutic information gathered during the total time spent on hd , were collected . the arithmetic mean of the monthly biochemical values since the beginning of hd until the date of beginning of the study was obtained . moreover , the cumulative doses of ca- , calcitriol- and al - containing phosphate binder received since the beginning of dialysis were also investigated . in order to diagnose vascular calcifications , all patients underwent x - ray studies ( thoracic , lumbar spine , pelvic and hand ) . the analyses were done grouping the arteries into three types according to their size and the predominant constituent of the tunica media : large or elastic arteries ( aorta and iliac ) in which elastic fibres are the main component of the media ; medium ( muscular or distributive ) arteries ( femoral , uterine / spermatic and radial ) in which smooth muscle is the predominant constituent of the media and small arteries ( palmar arch and digital ) in which also a minor quantity of smooth muscle is present . abdominal aortic calcifications were graded according to their severity into mild ( isolated punctiform calcifications ) , moderate ( linear calcifications with a maximum length of two vertebral bodies or one dense plaque ) and severe ( linear calcifications with a length of two or more vertebral bodies and/or two or more dense plaques ) . iliac , femoral , uterine / spermatic , radial , palmar arch and digital vascular calcifications were also graded and grouped as mild moderate when non - confluent calcifications or the partial section of the vessel was calcified , and severe if contiguous calcifications involving multiple segments or the whole section of the vessel were calcified . thoracic and lumbar spine x - rays were used to diagnose vertebral fractures by a standard procedure detailed elsewhere . vertebral fractures were classified according to genant 's semiquantitative method , which considers osteoporotic vertebral fractures as a reduction of the vertebral height ( anterior , posterior or middle ) > 20% after excluding other causes of vertebral deformities . non - vertebral ( peripheral ) osteoporotic fractures were included in the analysis only if they had been previously confirmed in the medical records . fractures were defined as osteoporotic when they fulfilled the next two criteria : ( a ) they were caused by a minor trauma or by a fall from a maximum height equal to or below the upright position of the patient and ( b ) localized in typical areas of osteoporotic fractures excluding skull , hands and feet . vascular calcifications and vertebral fractures were blindly evaluated by two independent experts with an inter - observer concordance of 90% and a kappa index of 0.73 . the hd patients were prospectively followed up for 2 years ; patients contributed as person - time until they underwent kidney transplantation , reached the end of the follow - up period , died or were lost to follow - up . the prevalences of aortic calcifications and vertebral fractures were compared to a random - based general population cohort from the same geographic area ( 308 men and 316 women ; mean age 65 9 years ) who participated in the european vertebral osteoporosis study ( evos ) , in which x - ray evaluations of thoracic and lumbar spine were carried out following the aforementioned standard procedure . the clinical , biochemical and therapeutic data gathered during the total time spent on hd were as follows : general and clinical data : gender , date of birth , profession , centre of dialysis , height , weight , dialysis treatment , dairy product intake after starting dialysis , physical activity , age of menarche , age of menopause , number of births , breastfeeding , hysterectomy and peripheral and vertebral fractures.renal disease and general hd data : aetiology of renal disease ( nephrosclerosis and/or ischaemic renal disease , diabetes , polycystic kidney disease , glomerular disease , tubulointersticial disease , other aetiology , unknown aetiology ) , total time on dialysis , age of starting dialysis , number of renal transplants , time on transplant , time on replacement therapy ( the sum of time on hd , time on peritoneal dialysis and time on transplant ) , localization of functioning arterious venous fistula , time of each hd session , bath solution use in a hd session ( bicarbonate , ca acetate ) , kt / v and pcr.risk factors and background : diabetes , time on diabetes , alcohol habits ( actual hard - drinking , non - actual hard - drinking , no hard - drinking ) , tobacco habits ( current smoker , non - smoker , former smoker ) , high blood pressure , years of high blood pressure , bone diseases , endocrine diseases , neurological diseases , gastroenterologic diseases , respiratory diseases , cardiovascular diseases , urologic diseases , parathyroidectomy and age of parathyroidectomy.biochemical data : serum ca , p , ca p product , parathyroid hormone ( pth ) , alkaline phosphatase , al , total cholesterol , hdl - cholesterol , triglycerides , total proteins , albumin , haemoglobin , haematocrit and bicarbonate.treatment : cumulative dose of ca- , calcitriol- and al - containing compounds , corticosteroids , anticoagulants , thyroid hormones , lipid - lowering drugs and insulin . general and clinical data : gender , date of birth , profession , centre of dialysis , height , weight , dialysis treatment , dairy product intake after starting dialysis , physical activity , age of menarche , age of menopause , number of births , breastfeeding , hysterectomy and peripheral and vertebral fractures . renal disease and general hd data : aetiology of renal disease ( nephrosclerosis and/or ischaemic renal disease , diabetes , polycystic kidney disease , glomerular disease , tubulointersticial disease , other aetiology , unknown aetiology ) , total time on dialysis , age of starting dialysis , number of renal transplants , time on transplant , time on replacement therapy ( the sum of time on hd , time on peritoneal dialysis and time on transplant ) , localization of functioning arterious venous fistula , time of each hd session , bath solution use in a hd session ( bicarbonate , ca acetate ) , kt / v and pcr . risk factors and background : diabetes , time on diabetes , alcohol habits ( actual hard - drinking , non - actual hard - drinking , no hard - drinking ) , tobacco habits ( current smoker , non - smoker , former smoker ) , high blood pressure , years of high blood pressure , bone diseases , endocrine diseases , neurological diseases , gastroenterologic diseases , respiratory diseases , cardiovascular diseases , urologic diseases , parathyroidectomy and age of parathyroidectomy . biochemical data : serum ca , p , ca p product , parathyroid hormone ( pth ) , alkaline phosphatase , al , total cholesterol , hdl - cholesterol , triglycerides , total proteins , albumin , haemoglobin , haematocrit and bicarbonate . treatment : cumulative dose of ca- , calcitriol- and al - containing compounds , corticosteroids , anticoagulants , thyroid hormones , lipid - lowering drugs and insulin . informed consent was obtained from all the recruited patients and approved by the clinical research ethics committee of asturias . the prevalences of vascular calcifications and vertebral fractures were expressed as percentages with 95% confidence intervals ( ci ) . differences in clinical and biochemical data and the association between clinical and biochemical variables and vascular calcifications were compared using t - test analyses , multivariable logistic regression analyses and also non - parametric tests ( mann whitney u - test ) if required . the strength of the association between the prevalence of vascular calcifications and the prevalence of vertebral fractures was compared by calculating the or and 95% ci using multivariable logistic regression analyses . all these analyses were adjusted by age , time on dialysis , time on rrt , haemoglobin , diabetes , sex and all treatments administered . cox multiple regression analyses for censored survival data were performed to compare survival between subjects with and without prevalent vertebral fractures and vascular calcifications . the analyses were performed separately for men and women after adjusting by age and time on dialysis . the main clinical and biochemical data collected are summarized in table 1 ; there were no statistically significant differences between the sexes . clinical variables in hd patients by sex rrt , renal replacement therapy ; hd , haemodialysis ; pth , parathyroid hormone . ipth and cumulative intake of ca , calcitriol and al were expressed as median and interquartile range . in this case , the mann based on the calibre of the arteries , the prevalence of vascular calcifications was 86.4% in large calibre , 70.5% in medium calibre and 20.2% in small calibre arteries . the prevalence of aortic calcifications was higher in hd patients than in the general population ( 79% versus 37.5% , p < 0.001 ) . this difference was observed in both sexes ( 77.8% versus 29.7% in women , p < 0.001 and 80.3% versus 45.4% in men , p < 0.001 ) . in hd patients , the or for having prevalent aortic calcifications ( all grades ) compared with the general population was 8.7 ( 5.015.0 ) . in women , the or was slightly higher than that in men [ or = 9 ( 3.821 ) and or = 7.7 ( 3.716.1 ) , respectively ] . mean serum ca , p , ca p , median ipth and the median cumulative intake of calcitriol- , ca- or al - containing phosphate binders , corticosteroids and other therapies were not significantly associated with the prevalence of any type of vascular calcifications ( table 2 ) . from the rest of the factors analysed , only age , time on hd , total time on any rrt ( hd , peritoneal dialysis or renal transplantation ) and diabetes were positively and significantly associated with a higher prevalence of vascular calcifications ( table 3 ) . after age and sex adjustments , the multivariate logistic regression analysis showed that only time on rrt and diabetes maintained the positive association . mean and standard deviation values of biochemical parameters ( ca , p and pth ) , median values and interquartile range of pth , and cumulative dose of calcitriol , ca and al in patients with ( yes ) or without ( no ) vascular calcifications in different arterial localizations pth , parathyroid hormone . age , time on haemodialysis ( hd ) , time on renal replacement therapy ( rrt ) in patients with ( yes ) or without ( no ) vascular calcifications at different calibre arteries significant difference , p < 0.05 . age was associated with vascular calcifications in large and medium calibre arteries while time on hd and total time on rrt were associated with an increased risk of vascular calcification , only in medium calibre arteries , such as femoral arteries [ or = 1.26 ( 1.071.48 ) and or = 1.17 ( 1.041.32 ) ] , radial arteries [ or = 1.17 ( 1.021.33 ) and or = 1.13 ( 1.021.24 ) ] and uterine / spermatic arteries [ or = 1.20 ( 1.041.39 ) and or = 1.15 ( 1.031.29 ) ] . diabetes was the most relevant variable associated with an increased risk of vascular calcifications , mainly in small and medium calibre arteries , such as digital [ or = 18.2 ( 4.770.2 ) ] , palmar arch [ or = 17.6 ( 4.865 ) ] , radial [ or = 13.4 ( 3.847.7 ) ] , uterine / spermatic arteries [ or = 3.1 ( 1.19.1 ) ] and femoral arteries [ or = 5.5 ( 1.618.5 ) ] , but no association was found in large calibre arteries , iliac and aorta arteries [ or = 0.4 ( 0.11.5 ) and or = 1.1 ( 0.33.9 ) , respectively ] . the other multiple clinical conditions detailed before showed no association with vascular calcifications at any site in the univariate and multivariate analyses . the prevalence of severe vascular calcifications was 63.6% and 70.7% in large calibre arteries , 55.6% in medium calibre arteries and 14.1% in small calibre arteries . the severity of vascular calcifications in small calibre arteries was significantly higher in men than in women ( 20.3% versus 5% , p < 0.05 ) . there was no association between the severity of vascular calcifications and biochemical parameters and the cumulative dose of drugs received . in large and medium calibre arteries , higher haemoglobin levels were associated with a lower prevalence of severe vascular calcifications [ or = 0.74 ( 0.550.99 ) and or = 0.65 ( 0.440.96 ) , respectively ] . among the other registered and analysed factors , time on rrt was positively and significantly associated with the prevalence of severe vascular calcifications in large and medium calibre arteries [ or = 1.23 ( 1.031.47 ) and or = 1.18 ( 1.021.36 ) , respectively ] ; the time on hd was also positively and significantly associated with the prevalence of severe vascular calcifications in medium calibre arteries [ or = 1.47 ( 1.151.87 ) ] . similarly , diabetes was also positively associated with the severity of vascular calcifications [ or = 6.43 ( 1.6525.11 ) ] . this effect was more remarkable in small and medium calibre arteries compared with large calibre arteries [ small calibre : or = 83.17 ( 11.81585.86 ) , medium calibre : or = 33.38 ( 3.69301.88 ) and large calibre : or = 2.07 ( 0.577.54 ) ] . the prevalence of vertebral fractures in hd patients was 26.5% , slightly higher in women than in men ( 33.3% versus 21.7% , p = 0.162 ) and similar to the values found in the general population , 24.1% ( 27.3% in women versus 20.7% in men , it was more common to have two or more vertebral fractures in women than in men ( 25.5% versus 6% , p < 0.05 ) . among all the different clinical and biochemical parameters analysed , only age and time on hd showed a significant , positive association with the prevalence of vertebral fractures . the prevalence of non - vertebral osteoporotic fractures in hd patients was 13.5% ( 26 fractures in total : 12 forearms , 6 hip fractures and 8 at other sites ) . among all the different variables analysed ( general , clinical , biochemical and therapeutic ) , only age was positively associated with the prevalence of peripheral osteoporotic fractures . vascular calcifications in medium calibre arteries were statistically significant and positively associated with a higher rate of prevalent vertebral fractures , but there was no relationship between vascular calcifications and peripheral osteoporotic fractures . at other sites such as large calibre arteries , there was a trend , but no statistically significant differences ( table 4 ) . relative risk ( or ) of vertebral fractures in haemodialysis patients with ( yes ) or without ( no ) vascular calcifications in different calibre arteries , adjusted by age and sex significant difference , p < 0.05 . after 2 years of follow - up , 25 women ( 34.7% ) , and 39 men ( 32.2% ) died . in men in contrast , 93.3% of women who died had severe vascular calcifications at least in one of the studied areas . in women ( figure 1a ) , severe vascular calcifications were positively associated with mortality after adjustments by age and time on hd ( cox multiple regression analyses ) [ rr = 3.2 ( 1.010.0 ) , p < 0.05 ] . when serum ipth , serum ca and cumulative intake of calcitriol were included as covariates , a similar behaviour was observed [ rr = 3.8 ( 1.113.6 ) , p < 0.05 ] , but only severe vascular calcifications were independently associated with mortality . meier analysis in women ( a ) with prevalent severe vascular calcifications at any vascular site , as figure 1b depicts , women with vertebral fractures showed also a higher mortality rate [ rr = 4.8 ( 1.713.4 ) ] adjusted by age , time on hd and severe vascular calcifications ( cox multiple regression analysis ) ] . when serum ipth , serum ca and cumulative intake of calcitriol were included as covariates , a more marked effect was observed [ rr = 6.3 ( 2.020.3 , p < 0.005 ) , but only the prevalence of vertebral fracture was independently associated with mortality . no relationship between non - vertebral fractures and mortality was found either in men or in women . based on the calibre of the arteries , the prevalence of vascular calcifications was 86.4% in large calibre , 70.5% in medium calibre and 20.2% in small calibre arteries . the prevalence of aortic calcifications was higher in hd patients than in the general population ( 79% versus 37.5% , p this difference was observed in both sexes ( 77.8% versus 29.7% in women , p < 0.001 and 80.3% versus 45.4% in men , p < 0.001 ) . in hd patients , the or for having prevalent aortic calcifications ( all grades ) compared with the general population was 8.7 ( 5.015.0 ) . in women , the or was slightly higher than that in men [ or = 9 ( 3.821 ) and or = 7.7 ( 3.716.1 ) , respectively ] . mean serum ca , p , ca p , median ipth and the median cumulative intake of calcitriol- , ca- or al - containing phosphate binders , corticosteroids and other therapies were not significantly associated with the prevalence of any type of vascular calcifications ( table 2 ) . from the rest of the factors analysed , only age , time on hd , total time on any rrt ( hd , peritoneal dialysis or renal transplantation ) and diabetes were positively and significantly associated with a higher prevalence of vascular calcifications ( table 3 ) . after age and sex adjustments , the multivariate logistic regression analysis showed that only time on rrt and diabetes maintained the positive association . mean and standard deviation values of biochemical parameters ( ca , p and pth ) , median values and interquartile range of pth , and cumulative dose of calcitriol , ca and al in patients with ( yes ) or without ( no ) vascular calcifications in different arterial localizations pth , parathyroid hormone . age , time on haemodialysis ( hd ) , time on renal replacement therapy ( rrt ) in patients with ( yes ) or without ( no ) vascular calcifications at different calibre arteries significant difference , p < 0.05 . age was associated with vascular calcifications in large and medium calibre arteries while time on hd and total time on rrt were associated with an increased risk of vascular calcification , only in medium calibre arteries , such as femoral arteries [ or = 1.26 ( 1.071.48 ) and or = 1.17 ( 1.041.32 ) ] , radial arteries [ or = 1.17 ( 1.021.33 ) and or = 1.13 ( 1.021.24 ) ] and uterine / spermatic arteries [ or = 1.20 ( 1.041.39 ) and or = 1.15 ( 1.031.29 ) ] . diabetes was the most relevant variable associated with an increased risk of vascular calcifications , mainly in small and medium calibre arteries , such as digital [ or = 18.2 ( 4.770.2 ) ] , palmar arch [ or = 17.6 ( 4.865 ) ] , radial [ or = 13.4 ( 3.847.7 ) ] , uterine / spermatic arteries [ or = 3.1 ( 1.19.1 ) ] and femoral arteries [ or = 5.5 ( 1.618.5 ) ] , but no association was found in large calibre arteries , iliac and aorta arteries [ or = 0.4 ( 0.11.5 ) and or = 1.1 ( 0.33.9 ) , respectively ] . the other multiple clinical conditions detailed before showed no association with vascular calcifications at any site in the univariate and multivariate analyses . the prevalence of severe vascular calcifications was 63.6% and 70.7% in large calibre arteries , 55.6% in medium calibre arteries and 14.1% in small calibre arteries . the severity of vascular calcifications in small calibre arteries was significantly higher in men than in women ( 20.3% versus 5% , p < 0.05 ) . there was no association between the severity of vascular calcifications and biochemical parameters and the cumulative dose of drugs received . in large and medium calibre arteries , higher haemoglobin levels were associated with a lower prevalence of severe vascular calcifications [ or = 0.74 ( 0.550.99 ) and or = 0.65 ( 0.440.96 ) , respectively ] . among the other registered and analysed factors , time on rrt was positively and significantly associated with the prevalence of severe vascular calcifications in large and medium calibre arteries [ or = 1.23 ( 1.031.47 ) and or = 1.18 ( 1.021.36 ) , respectively ] ; the time on hd was also positively and significantly associated with the prevalence of severe vascular calcifications in medium calibre arteries [ or = 1.47 ( 1.151.87 ) ] . similarly , diabetes was also positively associated with the severity of vascular calcifications [ or = 6.43 ( 1.6525.11 ) ] . this effect was more remarkable in small and medium calibre arteries compared with large calibre arteries [ small calibre : or = 83.17 ( 11.81585.86 ) , medium calibre : or = 33.38 ( 3.69301.88 ) and large calibre : or = 2.07 ( 0.577.54 ) ] . the prevalence of vertebral fractures in hd patients was 26.5% , slightly higher in women than in men ( 33.3% versus 21.7% , p = 0.162 ) and similar to the values found in the general population , 24.1% ( 27.3% in women versus 20.7% in men , p = 0.056 ) . it was more common to have two or more vertebral fractures in women than in men ( 25.5% versus 6% , p < 0.05 ) . among all the different clinical and biochemical parameters analysed , only age and time on hd showed a significant , positive association with the prevalence of vertebral fractures . the prevalence of non - vertebral osteoporotic fractures in hd patients was 13.5% ( 26 fractures in total : 12 forearms , 6 hip fractures and 8 at other sites ) . among all the different variables analysed ( general , clinical , biochemical and therapeutic ) , only age was positively associated with the prevalence of peripheral osteoporotic fractures . vascular calcifications in medium calibre arteries were statistically significant and positively associated with a higher rate of prevalent vertebral fractures , but there was no relationship between vascular calcifications and peripheral osteoporotic fractures . at other sites such as large calibre arteries , there was a trend , but no statistically significant differences ( table 4 ) . relative risk ( or ) of vertebral fractures in haemodialysis patients with ( yes ) or without ( no ) vascular calcifications in different calibre arteries , adjusted by age and sex significant difference , p < 0.05 . after 2 years of follow - up , 25 women ( 34.7% ) , and 39 men ( 32.2% ) died . in men , in contrast , 93.3% of women who died had severe vascular calcifications at least in one of the studied areas . in women ( figure 1a ) , severe vascular calcifications were positively associated with mortality after adjustments by age and time on hd ( cox multiple regression analyses ) [ rr = 3.2 ( 1.010.0 ) , p < 0.05 ] . when serum ipth , serum ca and cumulative intake of calcitriol were included as covariates , a similar behaviour was observed [ rr = 3.8 ( 1.113.6 ) , p < 0.05 ] , but only severe vascular calcifications were independently associated with mortality . kaplan meier analysis in women ( a ) with prevalent severe vascular calcifications at any vascular site , ( b ) with prevalent vertebral fractures . as figure 1b depicts , women with vertebral fractures showed also a higher mortality rate [ rr = 4.8 ( 1.713.4 ) ] adjusted by age , time on hd and severe vascular calcifications ( cox multiple regression analysis ) ] . when serum ipth , serum ca and cumulative intake of calcitriol were included as covariates , a more marked effect was observed [ rr = 6.3 ( 2.020.3 , p < 0.005 ) , but only the prevalence of vertebral fracture was independently associated with mortality . no differences were found in men . no relationship between non - vertebral fractures and mortality in the recent years , the study of vascular calcifications , demographic , clinical and biochemical parameters and bone fractures in patients with ckd has gained great interest , mainly due to their great impact on morbidity and mortality . calcification in the vessel walls occurs at two sites : the intima and the media . calcification of the intima is frequently localized in the aorta and coronaries and associated with atherosclerotic burden . calcification of the media occurs in the elastic lamina of the large and medium calibre arteries , it increases with age and it is frequent in ckd and diabetic patients . the complications of these two types of vascular calcifications are different ; however , they greatly account for the increase in morbidity and mortality of ckd patients [ 2729 ] . in this study , prevalent aortic calcifications were significantly higher in hd patients ( 79% ) than in a random - based general population of the same age , sex and region ( 37.5% ) . moreover , women on hd showed an increased risk of having severe aortic calcifications compared with women from the general population , probably due to a combination of atherosclerosis and arteriosclerosis with intima and media vascular calcifications . most of the previous studies have focused their interest on the aorta and coronary arteries . in this study , however , we also looked at other sites ( digital , palmar arch , radial , uterine / spermatic , femoral and iliac arteries ) and we also looked at the severity of vascular calcifications using a semiquantitative method . another original and important aspect of the present work was to investigate the relationship between the prevalence of vascular calcifications in different arterial territories classified according to their size and the predominant constituent of tunica media and the different variables studied [ 3133 ] . we found a relationship between the prevalence of vascular calcifications and the calibre of the arteries . in hd patients , the prevalence of vascular calcifications in large calibre arteries was higher than that in medium and small calibre arteries-four times in the latter . these results could be at least partly due to the different arterial wall structure of the three categories of arteries studied , which may also imply relevant functional changes in the vasculature . in addition , the severity of vascular calcifications in small calibre arteries was significantly higher in men than in women ( 19.4% versus 4.8% , p < 0.05 ) , a finding that could be partly related to the protective role of oestrogens in inflammation , atherosclerosis and calcification , a common finding in young women but also verifiable even in elderly women [ 3537 ] . among demographic , general and clinical parameters , age was positively associated with vascular calcifications in large and medium calibre arteries , reinforcing the role of age as a risk factor for atherosclerosis and arteriosclerosis . time on hd and total time on rrt were positively associated with vascular calcifications , particularly in medium calibre arteries , in which each year on rrt increased the risk of having vascular calcifications by 15% . these results are in agreement with previous studies that have identified the time spent on dialysis as an important risk factor , mainly for the medial but also for the intima arterial calcifications . other meaningful clinical or biochemical variables , such as hypertension , hyperlipidaemia , hypercalcaemia , hyperphosphataemia , hyper- or hypoparathyroidism , corticosteroid treatment , cumulative intake of ca- , calcitriol- and al - containing phosphate binders or elevated ca p product , did not show any significant association with the prevalence and severity of vascular calcifications . interestingly , high serum haemoglobin levels were associated with a lower prevalence of severe vascular calcifications in large and medium calibre arteries . this finding is in agreement with the well - known fact that chronic anaemia results in an increased cardiac output which , over time , leads to ventricular dilation , hypertrophy and the arterial remodelling of central elastic arteries such as the aorta and the carotids , leading to arterial enlargement , compensatory arterial intima - media thickening and arteriosclerosis . diabetes , a highly prevalent condition in hd patients , was strongly associated with the prevalence of calcifications , particularly in small calibre arteries . in fact , the risk of having any type of vascular calcification increased from 18 to 38 times in all the comparisons carried out between diabetic and non - diabetic hd patients . this strong , positive association has been already reported in diabetic patients with no ckd [ 4143 ] . so far , few studies on hd patients have focused on the prevalence of vertebral fractures , clinical and biochemical parameters and current treatments . we found that the prevalence of vertebral fractures ( 26.5% ) was higher than that documented in previous studies [ 4446 ] , but similar to that observed in a general population ( 25% ) recruited in the same geographical area with similar age and sex patterns to those of the cohort of hd patients studied . only age and time on hd showed a positive and statistically significant association with a higher prevalence of vertebral fractures . in agreement with other recently published studies , none of the biochemical variables or previous treatments showed any significant association with vertebral fractures . non - vertebral ( peripheral ) osteoporotic bone fractures , particularly hip fractures , seem to be more prevalent in dialysis patients . in agreement with the dopps ii study ( 2.6% ) , we observed a 3% prevalence of hip fractures and a clearly positive association of hip fractures with age . although pth levels might be an independent risk factor for osteoporotic fractures , the association of these two parameters is difficult to prove in cross - sectional or short - term follow - up studies ( 2 years ) , as is the case in our study . did not find any association between pth and hip fracture in hd patients . in any case , the small number of hip fractures ( n = 6 ) in our study might be insufficient to infer a more detailed explanation . even though the pathogenetic factors linking vascular calcifications and bone fragility are not clear , several studies have shown a greater predisposition to develop vascular calcifications when bone turnover is low [ 5052 ] . in our study , we have found a positive relationship between osteoporotic vertebral fractures and vascular calcifications in some large and medium calibre arteries , mainly in femoral and uterine / spermatic , but not in small calibre arteries . this finding could be related to the reduced amount of muscle cells present in small calibre arteries . in fact , only vascular smooth cells derived from the media from large and medium - size calibre arteries are those that undergo calcification and bone- and cartilage - like phenotypic changes under several in vitro experimental conditions . in addition , the reduced number of patients with calcification in small calibre arteries makes it difficult to draw definitive and more solid conclusions in this area . in agreement with our results , other authors have also found a relationship between large - size arteries ( aorta ) and osteoporotic fractures in the general population , reinforcing the idea that there is a positive association between vascular calcifications and osteoporotic fractures in both the general and hd population . the framminghan study , carried out in the general population , has shown that vascular calcifications are independent predictors of vascular morbidity and mortality . in our study , only women on hd showed a positive association between prevalent severe vascular calcifications and mortality . conversely , no association was found in men , supporting the hypothesis that there are relevant sex differences in which the arterial remodelling effect of hormones could be implicated . cardiovascular risk factors may have a different impact according to sex ; for example , in the general population , cholesterol is more important for cardiovascular risk in men than in postmenopausal women , in whom hypertension , diabetes and its combination play a major role . similar to what has been described in the general population , hd women showed a positive association between prevalent vertebral fractures and mortality . this finding , after only 2 years of follow - up , stresses the role and importance of bone health as an independent parameter related not only to morbidity and quality of life but also to mortality . firstly , not only the relatively small sample size but also the only 2 years of follow - up may have prevented us from obtaining associations among some of the other relevant variables studied . secondly , since we used a non - highly sensitive technique such as x - rays to detect vascular calcifications , it is possible that the use of more specific and sensitive techniques , such as electron beam computed tomography ( ebct ) or spiral computed tomography [ 5760 ] , might have added additional useful information . however , in favour of our results , recent studies have shown a good correlation between ebct and x - rays . in any case , the fact that we found a positive association with a medium - size sample , using a less sensitive but widely available technique as the standardized x - rays , demonstrates the strength of the association between vascular calcifications , bone fractures and mortality . even though the pathogenetic factors linking vascular calcifications and bone fragility are not clear , several studies have shown a greater predisposition to develop vascular calcifications when bone turnover is low [ 5052 ] . in our study , we have found a positive relationship between osteoporotic vertebral fractures and vascular calcifications in some large and medium calibre arteries , mainly in femoral and uterine / spermatic , but not in small calibre arteries . this finding could be related to the reduced amount of muscle cells present in small calibre arteries . in fact , only vascular smooth cells derived from the media from large and medium - size calibre arteries are those that undergo calcification and bone- and cartilage - like phenotypic changes under several in vitro experimental conditions . in addition , the reduced number of patients with calcification in small calibre arteries makes it difficult to draw definitive and more solid conclusions in this area . in agreement with our results , other authors have also found a relationship between large - size arteries ( aorta ) and osteoporotic fractures in the general population , reinforcing the idea that there is a positive association between vascular calcifications and osteoporotic fractures in both the general and hd population . the framminghan study , carried out in the general population , has shown that vascular calcifications are independent predictors of vascular morbidity and mortality . in our study , only women on hd showed a positive association between prevalent severe vascular calcifications and mortality . conversely , no association was found in men , supporting the hypothesis that there are relevant sex differences in which the arterial remodelling effect of hormones could be implicated . cardiovascular risk factors may have a different impact according to sex ; for example , in the general population , cholesterol is more important for cardiovascular risk in men than in postmenopausal women , in whom hypertension , diabetes and its combination play a major role . similar to what has been described in the general population , hd women showed a positive association between prevalent vertebral fractures and mortality . this finding , after only 2 years of follow - up , stresses the role and importance of bone health as an independent parameter related not only to morbidity and quality of life but also to mortality . firstly , not only the relatively small sample size but also the only 2 years of follow - up may have prevented us from obtaining associations among some of the other relevant variables studied . secondly , since we used a non - highly sensitive technique such as x - rays to detect vascular calcifications , it is possible that the use of more specific and sensitive techniques , such as electron beam computed tomography ( ebct ) or spiral computed tomography [ 5760 ] , might have added additional useful information . however , in favour of our results , recent studies have shown a good correlation between ebct and x - rays . in any case , the fact that we found a positive association with a medium - size sample , using a less sensitive but widely available technique as the standardized x - rays , demonstrates the strength of the association between vascular calcifications , bone fractures and mortality .

phage infection is the most prevalent cause of decreased starter activity in cheese and fermented milks manufacturing , resulting in fermentations in which acid production is markedly reduced or , in extreme cases , blocked ( neve , 1996 ) . raw milk is considered to be the principal source of phages , either as free virions or as prophages present in wild strains of lactic acid bacteria ( lab ) and constitutes the primary phage entranceway to the industrial environment ( everson , 1991 ; josephsen and neve , 1998 ) . phages are difficult to eliminate because they rapidly disseminate in dairy plants ( neve et al . , 1989 ) . as phage infections lead to economic losses in dairy factories , the development of control measures becomes essential . this review is aimed to revise the information regarding the diverse treatments applied in dairy industry for sanitization and microbial inactivation , and their efficiency on the inactivation of bacteriophages present in the dairy environment . in dairy manufactures , raw milk is often subjected to a thermal treatment to eliminate the majority of microorganisms present , including spoilage and pathogens , thus assuring a good quality and longer shelf - life of the final product . many works have studied thermal resistance of lactic acid bacteriophages , especially focusing on temperatures traditionally used to sanitize the milk in the dairy industry ( 63 , 72 , and 90c ) , and on conditions recommended by the international dairy federation ( idf ; 90c for 15 min ) to guarantee complete phage inactivation ( svensson and christiansson , 1991 ) . for cheese production , low temperature long time ( ltlt , 63c for 30 min ) or high temperature short time ( htst , 72c for 15 s ) pasteurization were traditionally applied . ultra - high - temperature ( uht ) processing , also called ultra - pasteurization ( more than 135c for 12 s ) , is a sterilization treatment as it produces spores destruction . however , this high heating can cause maillard browning and negatively affect the taste and smell of dairy products . milk used for yogurt production is generally treated at 80c for 30 min or at 95c for 10 min ( soukoulis et al . , 2007 ) . regardless of the high efficiency of this heat treatment to inactivate microorganisms , it is not always effective against dairy bacteriophages . the effects of such treatments were monitored by transmission electron microscopy on a heat - sensitive and on a heat - resistant lactococcus lactis phage by atamer et al . release of phage dna from viral capsids , decomposition of phage into head and tail structures , and aggregation of phage tails were the most frequently seen phenomena , principally for the heat - sensitive phage studied . for the heat - resistant one , these authors concluded that heat resistance is associated with both increased stability of viral dna packaged in the capsids and enhanced stability of phage particles as a whole . one of the most used is t99 , defined as the time to achieve 99% inactivation of phage particles , which allows having a very good approximation of phage resistance against inactivation treatments . however , this parameter does not consider total phage destruction , but inactivation of the most sensitive 99% of phage population . whether a heat treatment is applied to samples containing elevated initial phage loads , remaining viral particles can multiply and reach problematic levels again . with this regard , the minimum time necessary for complete phage inactivation ( considering it when phage counts this time may be much longer than the corresponding t99 and , ideally , both parameters should be determined . table 1 overviewed the susceptibility to 63 , 72 , and 90c treatments ( expressed as t99 values ) of some bacteriophages from lactic acid / probiotic bacteria of great importance in fermentative dairy industry . regardless the thermal treatment applied , a great diversity in heat susceptibility has been observed . consequently , thermal resistance would depend more on the particular phage studied than on the species specificity . for instance , some bacteriophages specific of l. lactis and streptococcus thermophilus were rapidly inactivated at 63c ( t99 values from 2.7 to 12 min ) , whereas some other phages infecting these species revealed high resistance to the same or even harsher treatments ( binetti and reinheimer , 2000 ; surez and reinheimer , 2002 ) . low thermal resistance is not confined to phages infecting dairy cocci ( l. lactis and s. thermophilus ) ; a variety of lactobacillus phages with an extremely heat sensitivity was also discovered . among the latter , it is worth noticing phages hv ( lactobacillus helveticus ) , cb1/204 ( lactobacillus delbrueckii ) , pl-1 and j-1 ( lactobacillus casei and lactobacillus paracasei ) , which showed very low t99 values ( from 2.1 to 3.1 min ) in all suspension media tested ( quiberoni et al . , 1999 ; capra et al . , 2004 ; ebrecht et al . , 2010 ) . in spite of the high heat sensitivity to 63c shown by some lactobacillus phages , there were others capable to easily resist this treatment , as demonstrated by lactobacillus plantarum phages investigated by briggiler marc et al . susceptibility of dairy phages to heat treatments , using reconstituted skim milk as suspension media . . as it can be deduced , increasing the temperature from 63 to 72c improved the efficiency of phage inactivation . good examples are phages 001 ( l. lactis ) , 0bj and cym ( s. thermophilus ) , cnrz 832-b1 ( lb . plantarum ) , showing high t99 values after incubation at 72c . sometimes , even if t99 values were not so elevated ( e.g. , 2.5 min for phage ib3 ) , the time to destroy the total viral population was much longer ( > 45 min ) . in these cases , inactivation curves might be fitted into two or even three independent linear components ( daoust et al . , 1965 ) . attempts to explain this behavior suggested that a mixture of phage particles with different heat sensitivity might exist : a rapidly inactivated population , responsible for the low t99 values obtained , and a heat - resistant one , explaining the long time required for total phage inactivation . both populations would follow a first - order reaction , but with a decreased slope for the latter ( hiatt , 1964 ) . it has been suggested that heat - resistant phages naturally occur in all types of dairy environments as a low proportion of the whole phage population ( atamer et al . , 2009 ) . this fact remarks the importance of consider both parameters when defining the heat sensitivity of a given phage . similarly to phage ib3 , destruction of total viral populations for the above mentioned heat - resistant phages was generally not achieved at this temperature , since detectable viable phage particles were found even after 45 min of treatment , which is a much longer period than that normally applied in the dairy industry for milk sanitization ( quiberoni et al . , 1999 , 2003 ; binetti and reinheimer , 2000 ; surez and reinheimer , 2002 ; briggiler marc et al . , 2009 ) . dissimilar susceptibility to short - time pasteurization conditions ( 72c for 30 s ) was found for two lactococcus phages ; whereas one of them was complete and rapidly inactivated ( loss of 7 log orders ) , the other one was highly heat - resistant ( reduction of 2 log orders ) and remained viable even after 60 min of treatment ( mller - merbach et al . , 2005 ) . figure 1 shows thermal inactivation kinetics at 72c of some dairy bacteriophages , when reconstituted skim milk ( rsm ) was used as suspension media . thermal destruction kinetics of phages ib3 ( ) , ll - h ( ) ( lb . delbruekii ) , fagk1 ( ) , atcc 8014-b1 ( ) ( lb . plantarum ) , 021 - 5 ( ) , and 0bj ( * ) ( s. thermophilus ) , at 72c using reconstituted skim milk as suspension media . temperatures higher than 7275c allowed efficient phage inactivation in a short time . since milk destined to yogurt manufacture is usually treated at 82c for 5 min , some authors have subjected lb . delbrueckii phage particles to this temperature ( ebrecht et al . , 2010 ) , finding total phage destruction within 2 min of treatment . p008 to 80c , demonstrating undetectable counts after only 1 min at this temperature . on the contrary , 40% of l. lactis phages tested were able to resist 80c for 5 min ( atamer et al . , 2009 ) , whereas 70% of leuconostoc phages particles were still detectable after 1 min at this temperature ( atamer et al . , 2011 ) . until recently , heat treatment at 90c for 5 min was sufficient for inactivation of phage particles ( binetti and reinheimer , 2000 ; surez and reinheimer , 2002 ; capra et al . , 2004 ; briggiler marc et al . , 2009 ) . despite this , in the last years some extraordinarily heat - resistant phages are being found in many research laboratories , such as phage ib3 ( lb . delbrueckii ) , whose complete inactivation required at least 15 min at 90c ( quiberoni et al . , 2003 ) . moreover , some lactococcal phages were able to survive 5 min at 90c ( buzrul et al . ( 2009 ) revealed that phage p1532 was still detectable even after heating at either 90c for 20 min or 97c for 5 min . the protocol used for microbiological detection of phages in dairy samples includes a heat treatment at 90c for 15 min of control samples to be tested for non - phage inhibiting agents ( svensson and christiansson , 1991 ) . however , this standard protocol might not be appropriate for inactivating high heat - resistant phages ( atamer et al . , 2009 ) . furthermore , the number of phages with exceptional thermo - resistance seems to be increasing every day , which led to the need of revising the methodology applied to industrial samples suspected of containing phages . in some cases , milk seemed to be protective to viral particles , as observed for those of lb . similar conclusions were obtained by other authors when testing either lactococcus or leuconostoc phages ( atamer et al . the reason for the protective effect of milk is still being investigated ; however , it was postulated that certain components , especially casein , could be responsible for the increased thermal resistance of dairy phages . unlike protein , fat was found to have no influence on phage inactivation ( atamer et al . , 2010 ) . for example , surez and reinheimer ( 2002 ) determined that phosphate buffer was the most protective medium in comparison with m17-broth and skim milk . sometimes , it was not possible to establish a clear influence of the suspension media . helveticus phages , for example , resisted better in mrs broth , but others did it in tmg buffer ( quiberoni et al . , 1999 ) . likewise , not conclusive influence was found by capra et al . a biocide should fulfill several criteria to be usable in the food industry , for example possessing a fast antimicrobial activity , ease of application , low cost , lack of negative impact on the final product , and degradation into harmless final products ( nicholds and wolf , 1945 ; schrder , 1984 ) . however , inactivation of bacteriophages was taken into consideration only recently as a criterion of biocides selection , which is reflected by an increasing number of studies directed to quantify their effectiveness in this sense . many of these works were carried out in our laboratory and biocides tested were , until recently , almost exclusively ethyl alcohol ( ethanol ) , isopropyl alcohol ( isopropanol ) , sodium hypochlorite , and peracetic acid , all of which will be further discussed . table 2 summarizes sensitivity ( expressed as t99 ) of phages of diverse bacterial species to ethanol and sodium hypochlorite used at different concentration . time ( min ) necessary to reach 99% inactivation ( t99 ) of phages infective for diverse species of lactic acid bacteria in presence of ethanol and sodium hypochlorite at different concentration . ethanol and , at much lesser extent , isopropanol , were tested on a broad spectrum of lab phages , at concentrations oscillating between 10 and 100% ( v / v ) , but the lower concentration did not produce in general any visible effect . thus , only 50% ( v / v ) or higher concentrations will be considered for discussion from now on . with respect to isopropanol , it was tested on phages of l. lactis ( surez and reinheimer , 2002 ) , s. thermophilus ( binetti and reinheimer , 2000 ) , lb . inactivation was almost undetectable and required very long incubation periods ( generally > 45 min ) , with only a few exceptions ( lactococcal phage 001 , lb . delbrueckii phage ll - h , and s. thermophilus phage 031-d ) where a milder effect was observed . streptococcus thermophilus phages cym , 021 - 4 , 021 - 5 , 0bj , and 031-d showed a moderate sensitivity to ethanol , as viral counts were 99% reduced ( t99 ) after less than 5 min of incubation in 75% ( v / v ) ethanol ( optimal concentration ) , and became undetectable after 15 min of contact with either 75 or 100% ( v / v ) concentrations . an exception was phage 021 - 4 , which greatly resisted 100% ( v / v ; > 45 min ; table 2 ) , although being highly sensitive to 75% ( v / v ) ethanol ( binetti and reinheimer , 2000 ) . lactococcal phages were , instead , more affected by pure ethanol than by 50 or 75% ( v / v ) dilutions , with the exception of phage qp4 , which possesses a very low resistance to 75% ( v / v ) ethanol . as a group , lb . collection phages cnrz 832-b1 and cnrz 0241 were inactivated by 50% ( v / v ) ethanol after 5 min of incubation . curiously , the former was moderately affected by a concentration of as low as 10% ( v / v ) ethanol , being the only phage with a reported t99 lower than 45 min for such concentration ( table 2 ) . phages hv and atcc 15807-b1 , instead , were more sensitive to a higher concentration ( 75% v / v ) ( quiberoni et al . , 1999 ) . delbrueckii phages , either of virulent ( quiberoni et al . , 2003 ) or temperate ( ebrecht et al . , 2010 ) nature , were most sensitive to 100% ( v / v ) ethanol , and showed dissimilar t99 values at this concentration , ranging from 22.2 min for phage ib3 ( the most resistant ) to 2 min or less for phages yab and temperate phage cb1/204 ( table 2 ) . in turn , ll - h was the only lb . delbrueckii phage completely inactivated after 30 min in 75% ( v / v ) ethanol ( quiberoni et al . , 2003 ) . interestingly , temperate phages cb1/204 and cb1/342 presented lower t99 values than 3 out of the 4 virulent phages studied ( table 2 ) . concerning either virulent phages pl-1 , j-1 ( capra et al . , 2004 ) , and mlc - a ( capra et al . , 2006 ) , or temperate phages ilp84 and ilp1308 ( mercanti et al . , 2012 ) , all infective for lb . paracasei , a similar pattern of inactivation was obtained after incubation with ethanol : a fast initial reduction of about two - log orders was followed by a plateau , yielding almost the same phage titers after 15 or 45 min of treatment . plantarum phages were only slightly inactivated with 100% ( v / v ; the best concentration ) or 50% ( v / v ) ethanol , and almost not affected by 75% ( v / v ) , which is optimal for many other lab phages ( briggiler marc et al . plantarum phages . with respect to the mechanism of action of ethanol , a study conducted by maillard et al . ( 1996a ) showed capsid alterations on f116 , a phage infective for pseudomonas aeruginosa ; as expected , no dna damage was evidenced . the antibacterial attribute of alcohols is mainly due to alterations on the lipid fraction of the plasmatic membrane . for this reason , naked phages are likely to be naturally more resistant than bacteria to ethanol challenging . to sum up , it can be concluded that there are three levels of phage sensitivity to ethanol . in general , the most susceptible ( though moderately ) phages were those infecting dairy cocci : l. lactis and s. thermophilus . among lactobacilli , in general , the efficiency of phage inactivation accomplished by ethanol , though superior than that of isopropanol , is still not sufficient for industrial applications . besides , the absence of an optimal ethanol concentration able to kill a broad range of dairy phages makes this compound unsuitable as an anti - phage strategy in the dairy industry . resistance to sodium hypochlorite of phages infective for diverse lab genera resembles that exhibited against ethanol . helveticus , l. lactis , and s. thermophilus phage particles were completely eliminated by a concentration of residual free chlorine as low as 100 ppm ( quiberoni et al . , 1999 ; binetti and reinheimer , 2000 ; surez and reinheimer , 2002 ) ; only l. lactis phages 046 and qp4 required a stronger inactivation treatment ( 300 ppm for 45 min and 200 ppm for 30 min , respectively ; surez and reinheimer , 2002 ) . on the other hand , two temperate lb . delbrueckii phages ( ebrecht et al . , 2010 ) were readily killed as well . however , likewise for ethanol inactivation , there were other lb . delbrueckii phages with a noticeably greater resistance ( bym , yab , and ll - h ) , requiring between 5 and 45 min in the presence of 400 ppm of residual free chlorine ( quiberoni et al . unexpectedly , it exhibited the highest phage resistance to sodium hypochlorite ever measured , requiring a minimal concentration of 1200 ppm of residual free chlorine for at least 45 min for complete inactivation ; this fact has been attributed to a long persistence of the phage in industrial environments ( quiberoni et al . , 2003 ) . plantarum phages , exposures of 15 min ( phage atcc 8014-b2 ) and 30 min ( phages atcc 8014-b1 , fagk1 , and fagk2 ) to 800 ppm of residual free chlorine were necessary to completely inactivate phage suspensions ( briggiler marc et al . , 2009 ) . ( 2004 ) reported a minimum incubation of 5 min at 800 ppm of residual free chlorine as necessary to completely inactivate lb . paracasei to another : a concentration of 400 ppm was able to quickly eliminate ilp1308 , but 30 min at 800 ppm were necessary for complete inactivation of ilp84 , even though a t99 < 2 min was calculated for the latter ( table 2 ) . as 800 ppm of free chlorine is a concentration perceptively higher than that permitted in the dairy industry ( briggiler marc et al . , 2009 ) , chlorine could be usable only at laboratory scale for the inactivation of lb . according to some studies , sodium hypochlorite would cause phage inactivation through aggregation of tail proteins ( maillard et al . , 1998 ) , in addition to several structural alterations to the bacteriophage capsid ; which is likely to be related to the release of phage dna into the external medium . overall , sodium hypochlorite could be regarded as a good ( but not excellent ) choice for industrial phage inactivation . it was quite effective for most challenged phages , but there are some of them which resisted biocide concentrations much greater than those permitted in an industrial sanitization process . peracetic acid ( also known as ethaneperoxoic acid or peroxyacetic acid ) is commercially available in the form of a mixture containing acetic acid , hydrogen peroxide , peracetic acid , and water in a quaternary equilibrium ( gehr et al . , 2003 ) . with regard to its use in the dairy industry its major advantages include its ability to efficiently inactivate most microorganisms , its resistance to catalase and peroxidase ( enzymes which break down hydrogen peroxide ) and its degradation into environmentally friendly products ( acetic acid and hydrogen peroxide ) . peracetic acid maintains its activity over a wide temperature ( 040c ) and ph ( 3.07.5 ) ranges and in hard water , and is not affected by protein residues . these characteristics allow to safely use it in the food industry and even for cleaning - in - place ( cip ) practices . concerning the virucidal activity of peracetic acid , there are many works reporting a fast and effective inactivation of a broad spectrum of lab phages , including phages infecting s. thermophilus ( binetti and reinheimer , 2000 ) , l. lactis ( surez and reinheimer , 2002 ) , and several species of dairy lactobacilli , including lb . in addition , this chemical proved to be effective for the inactivation of non - lab phages , such as p. aeruginosa phage f116 ( maillard et al . , 1993 ) . in all cases , a treatment of 5 min ( or 2 min in some cases ) at 40c was enough to drop phage counts to undetectable levels , even of phages highly resistant to other chemical or thermal treatments , such as lb . peracetic acid is normally used at a concentration of 0.15% ( v / v ) in water ; the low ph of this aqueous solution ( around 2 ) has been suggested to be responsible for phage inactivation ( mercanti et al . , schrder ( 1984 ) proposed 40c as the most effective temperature to inactivate bacteria , yeasts , and fungi using peracetic acid . plantarum ( briggiler marc et al . , 2009 ) , lb . casei and lb . paracasei ( mercanti et al . , 2012 ) phages was found to be similar at either 25 or 40c , thus recommending the use of this temperature for industrial convenience . peracetic acid exerts its action through the hydroxyl radical ( oh ) , which reacts with any oxidizable compound present in its neighborhood , damaging practically any kind of macromolecule associated with a microorganism and eventually leading to its death . complete rupture of the nucleic acid is accomplished during bacteriophage inactivation , but whether it occurs inside the capsid or after the dna is released due to a capsid structural damage remains unclear . such alteration of the capsid structure was observed by sds - polyacrylamide gel electrophoresis ( maillard et al . there are several new biocides that are being progressively used by the dairy industry . among these , it is worth mentioning : ( i ) quaternary ammonium chloride , ( ii ) peracetic and peroctanoic acids with hydrogen peroxide ( vortexx ; ecolab , 2011 ) , ( iii ) an alkaline chloride foam ( active cl ) , ( iv ) p - toluenesulfonchloroamide , sodium salt ( clorina f ; cibsa , 2011 ) , and ( v ) ethoxylated non - ylphenol with phosphoric acid . however , very scarce data about their efficiency on lab phages inactivation are available so far , with reports only on lb . both alkaline chloride foam or ethoxylated nonylphenol with phosphoric acid were exceptionally efficient , which could be attributable to their extreme ph values ( > 12 and < 2 , respectively ) ; phages of species were not detected after 2 min of treatment ( ebrecht et al . quaternary ammonium chloride proved to be a usable biocide as well , but its efficiency quickly diminished at concentrations slightly under the optimal recommended , especially for lb . paracasei phages were more sensitive to low concentrations of quaternary ammonium chloride , but also in this case its effectiveness differed among the two phages studied ( mercanti et al . , 2012 ) . on the contrary , p - toluensulfonchloroamide showed no reduction on phage numbers , even when this compound was utilized without dilution and for longer incubation times , up to 2 h on lb . delbrueckii phages studied were thoroughly inactivated by 0.13% ( v / v ) vortexx [ with the exception of phage ib3 , which was only inactivated by a concentration of 0.26% ( v / v ) ; ebrecht et al . , paracasei phage counts at a concentration of 0.26% ( v / v ) ( mercanti et al . , 2012 ) . vortexx is publicized as a novel product with excellent sanitizing results at room temperature and delivering wide antimicrobial activity , including yeasts , mold , and bacteria , at much lower concentration than peracetic acid used alone ( ecolab , 2011 ) . this lower peracetic acid concentration raises the ph of vortexx ( at the recommended concentration of use ) to values between 3.1 and 3.4 . unfortunately , this higher ph appears to be critical for the inactivation of some phages , as both ilp84 and ilp1308 were quick and completely inactivated by 0.15% ( v / v ) peracetic acid , whilst 0.26% ( v / v ) vortexx yielded no significant titer reductions after 20 min of incubation ( mercanti et al . , 2012 ) . raw milk is regarded as one of the main sources of phages , thus representing their most important way of entry to the industrial environment ( everson , 1991 ; josephsen and neve , 1998 ) . moreover , the capability of bacteriophages to remain in the air for long periods makes bioaerosols one of the most important dispersion routes of virions . unfortunately , none of the currently available technologies is appropriate to inactivate bacteriophages suspended in the air of dairies , either in laboratory or in processing plants ( quiberoni et al . , 1999 , 2004 , 2006 ) . the photocatalytic properties of tio2 have been the subject of much research , mainly for the photochemical pollutant oxidation . several advantages of photocatalysis make this methodology an excellent alternative to the traditional chemical disinfection : ( i ) absence of residues , ( ii ) simultaneous treatment of diverse pollutant mixtures , ( iii ) broad range , and ( iv ) ease of operation . besides , the low cost , high abundance and safety of tio2 make this compound the most frequently selected catalyst ( hoffmann et al . , 1995 ; fujishima et al . , 2000 ; de lasa et al . , 2005 ) . semiconductor tio2 generates highly oxidizing species ( o2-andoh ) when photoexcited by uv radiation , thus catalyzing various chemical reactions , including the decomposition of organic compounds and destruction of microorganisms ( horie et al . , 1996 ) . photocatalysis application has been mostly intended to destroy fungi , bacteria , and spores in the air ( kakita et al . , 2000 ; , 2001 ; koziumi and taya , 2002 ; khn et al . , 2003 ; cho et al . , 2005 ; pal et al . , 2007 ; ditta et al . , 2008 ; zacaras et al . , 2010 ) though its efficiency for the inactivation of viruses contained in bioaerosols has been scarcely explored . furthermore , only a very few studies have been focused on the combined application of uv and tio2 in order to avoid phage dissemination in the environment . sjogren and sierka ( 1994 ) were able to reduce the titer of a ms2 phage ( specific for escherichia coli ) suspension by a factor of 10 after 10 min of irradiation at 2 mw / m in the presence of tio2 in a continuously stirred batch reactor . ( 2000 ) have reported the inactivation of phage pl-1 ( l. casei ) by using a ceramic preparation coated with a mixture of oxides including tio2 and ago . the experiment was conducted in liquid medium and the suspended phages were subjected to irradiation with black - light ( bl ; 300400 nm ) in the presence of tio2 thin film . these authors demonstrated the conversion of the virions into inactivated ghost particles , having an empty head without dna . later , kashige et al . ( 2001 ) investigated the pl-1 phage inactivation mechanism ; the authors found that the genome dna inside the phage capsids was fragmented , suggesting a previous damage to the capsid protein by active oxygen species generated in the tio2 film - settled aqueous medium under bl . the generation of both superoxide anions ( o2- ) and hydroxyl radicals ( oh ) in the aqueous medium was verified by chemical analysis , whereas electrophoresis confirmed a considerable phage genome dna fragmentation . more recently , tio2 photocatalysis was reported as a feasible methodology for the inactivation of phages infecting several lab species , since this technology seemed to be very efficient to this purpose ( briggiler marc et al . , 2009 , 2011 ) . to our knowledge , these studies are the first ones referring photocatalysis ( uv radiation and tio2 ) applied to phages in bioaerosols . ( 2009 , 2011 ) consists of an uv - a radiation emitting system ( wavelength range of 300420 nm , maximum emission at about 350 nm ) , an irradiation chamber , and a support to hold the tio2-coated borosilicate glass plates ( zacaras et al . , 2010 ) . the photocatalytic inactivation was applied on phage suspensions ( 10 plaque forming units ( pfu)/ml ) prepared in distilled water . phage suspensions were taken in the tio2-coated plates , further covered with a borosilicate glass to create a thickness similar to bioaerosol drops , and finally irradiated in a chamber with uv light for a maximum period of 3 h. very dissimilar behaviors were demonstrated by each bacteriophage under the same photocatalytic treatment . pfu / ml ) were achieved for phage bym after 45 min of exposure , but 60 min were necessary to attain similar results ( < 50 pfu / ml ) for phages atcc 8014-b1 and atcc 8014-b2 . likewise , undetectable counts ( < 10 pfu / ml ) of phages j-1 and qf9 particles were reached after 120 min of exposure . contrarily , phages atcc 15807-b1 and ib3 were able to resist up to 180 min of treatment , exhibiting viability losses of only 1.2 log orders . a similar performance was demonstrated by phage mlc - a , with a loss of viable particles of only 2.4 log orders after 120 min of exposure , ( briggiler marc et al . , 2009 , 2011 ) . regarding the photocatalytic inactivation efficiency ( abs ) , values calculated by the authors differed in more than two - log orders among all the phages tested ( table 3 ) . the highest inactivation efficiency was experimented by the bacteriophage atcc 8014-b2 ( abs = 4.244 10 pfu / photon ) , whereas the lowest was that calculated for the phage cb1/342 ( abs = 0.171 10 pfu / photon ) . besides , the authors observed that related phages , lytic for the same strain and morphologically similar , evidenced different behavior under the same conditions of photocatalytic inactivation . attempting to explain these results , a dissimilar stability of viral proteins against the absorbed radiation energy considering the capsomers as a protective shield for viral dna against the damage caused by the photocatalysis , thus different primary and conformational structure of capsid proteins would be relevant factors affecting the resulting inactivation kinetics ( briggiler marc et al . , articles focusing on the photocatalytic inactivation of microorganisms and or viruses hardly report their efficiency in terms of photons absorbed . in this sense , the only known study concerning tio2 photocatalytic inactivation expressed in terms of photons absorbed was applied on bacterial cells , specifically on e. coli suspensions ( marugn et al . , 2008 ) and the kinetic analysis was reported . an apparent photonic efficiency in the order of 10 cfu / photon was calculated by the authors for the maximum e. coli disinfection rate achieved during the experiments . comparable inactivation orders ( of approximately 10 pfu / photon ) were obtained by briggiler marc et al . efficiency of photocatalytic inactivation of dairy bacteriophages ( data adapted from briggiler marc et al . , classification of the international committee on taxonomy of viruses ( ictv ) as illustrated by ackermann ( 2007 ) . briggiler marc et al . ( 2009 , 2011 ) demonstrated the efficiency of the photocatalysis as a new methodology to be applied in dairies , where lab represent the target of bacteriophage attacks . in comparison to the uv - c and uv - c / tio2 units currently available in the market for the environmental air purification ( kover synergy , 2010 ; novapure , 2010 ) , the uv - a radiation assayed by the authors has the advantage of safe use , thus allowing their application for long periods even in the presence of personnel . additional research , particularly focused on the photocatalysis inactivation mechanism and the method efficiency , are indispensable for the design of equipment to be used in industrial environments , either in plants or in laboratories . thermal treatments such as pasteurization and sterilization were traditionally used in food industry for assuring safety and longer shelf - life of food products . although they are efficient and economical processes to inactivate microorganisms , they can cause undesirable protein denaturation , non - enzymatic browning , and loss of vitamins and volatile flavor compounds . bearing in mind the idea of avoiding undesirable consequences of heating , in the last years considerable efforts were focused on the development of novel non - thermal preservation processes . among them , high - pressure processing is one of the most promising , because it combines maximal retention of the chemical and physicochemical product properties with efficient germ reduction ( lado and yousef , 2002 ; diels and michiels , 2006 ) . the most studied and applied pressure - based processes are high hydrostatic pressure ( hhp ) and high - pressure homogenization ( hph ) . although the apparent similarities between both technologies , they have some relevant differences that should be pointed out . first of all , they have very dissimilar exposure time ; this parameter abruptly diminishes from a few minutes in hhp to a second or less in hph ( wuytack et al . , 2002 ) . hph is a high hydrodynamic process during which the fluid is forced to pass through a small orifice and then subjected to an ultra - rapid decompression . the sudden fall in the local pressure of the fluid at constant temperature lead to the nucleation and growth of vapor bubbles ( or cavities ) within the body of liquid , the collapse of which could transmit several localized forces to surfaces or particles , including the microbial cell ( middelberg , 1995 ) . on the other hand , hhp systems consist of a pressure vessel , the pressure transmission fluid ( usually water ) and one or more pressurizing pumps . the packaged food is loaded into the vessel , the top is closed and the fluid is pumped into the vessel from the bottom . when the desired pressure is reached , pumping is stopped , valves are closed and the pressure is maintained without further need for energy input . the high pressure is applied isostatically so that all parts of the food are subjected to the same pressure at exactly the same time . in the case of liquids , such as fruit juices , the whole vessel can be filled with the juice , which becomes itself the pressure transmission fluid ( patterson , 2005 ) . homogenization was developed many years ago with the main purpose of enhancing the texture , taste , flavor , and shelf - life of food emulsions , particularly dairy products like milk and cream butter . as the consumers began to claim for more natural and fresh foods with improved stability , safety and extended shelf - life , a new generation homogenizers was consequently developed , giving as a result the origin of hph ( diels and michiels , 2006 ) . besides the food industry , hph is frequently applied in the pharmaceutical , cosmetic , and chemical industries for the preparation or stabilization of emulsions and suspensions , or to yield viscosity changes in products ( floury et al . , 2004 ) . hph - treated milk seemed to be very convenient to improve cheese quality and physicochemical properties of fermented milks as well ( burns et al . , hph could be also applied to cell disruption of yeasts and bacteria , thus causing a partial inactivation of the microbial population and allowing the release of intracellular components ( guerzoni et al . , 1999 ) . as a consequence , hph has been regarded as an alternative to other processes commonly applied for the inactivation of pathogenic and spoiling microorganisms ( heat , sterilization , and irradiation ) ( diels et al . , 2005 ; diels and michiels , 2006 ) . viral inactivation by hph treatments was not profoundly studied , especially when distinctively speaking of bacteriophages ( moroni et al . , 2002 ; diels and michiels , 2006 ; capra et al . 2009 ) . as it was previously mentioned , raw milk is regarded as the main source of phages entering to the dairies , some of them possessing a remarkable thermal resistance ( capra et al . , 2004 ; briggiler marc et al . , 2009 ) . nevertheless , only a few reports are available in the bibliography . by using electron microscopy , moroni et al . ( 2002 ) observed that hph - treated lactococcal phages were possibly inactivated by the loss of genetic material from fragmented phage heads . either total or partial loss of phage tails was also observed , rendering them incapable of adsorption to the bacterial cell wall . results obtained by the authors demonstrated a statistically significant difference in sensitivity to hph between the two morphology - types of phages : prolate - headed ( phage c2 ) and isometric - headed ( phages sk1 and ul36 ) , being the prolate head less stable than the isometric one . more recently , phages infecting several species of lactic and probiotic bacteria were also studied . amongst them , two temperate lb . paracasei phages ( ilp84 and ilp1308 ) showed a logarithmic reduction of viability along with the successive passes at 100 mpa when suspended in rsm ( mercanti et al . , 2012 ) . for these phages , falls of 4 and 4.5 log orders were observed , respectively , after five passes at 100 mpa ( figure 2 ) . these results were similar to those previously obtained for phages mlc - a ( specific for lb . helveticus ) subjected to the same experimental conditions ( capra et al . , 2009b ) , observing viability losses of 3.8 , 3.75 , and 2.0 log orders , respectively . in turn , phages mlc - a8 ( lb . casei ) , bym ( lb . delbrueckii ) , alq13.2 ( s. thermophilus ) , qp4 and qf12 ( l. lactis ) were not detected after only five passes at 100 mpa through the high - pressure homogenizer . it is interesting to emphasize that related phages mlc - a and mlc - a8 , lytic for the same probiotic lb . paracasei strain , showed remarkably different behavior after hph treatment , being the former more resistant than the latter . in addition , hph treatments applied to different initial concentrations of the same phage rendered dissimilar inactivation efficiency . contrarily to earlier reported results ( moroni et al . , 2002 ; capra et al . , 2009b ) , working specifically with phage mlc - a , found that the higher the initial phage load , the greater the reduction achieved . on the other hand , bacteriophage inactivation rate showed to be proportional to both applied pressure and number of passes . concerning the influence of suspension media ( milk , whey permeate , buffer ) , results were very dependent on each phage . although some authors ( moroni et al . , 2002 ) reported diminished hph effectiveness in milk and whey permeate , no clear protective effect for any of the assayed media was detected by capra et al . viability of dairy bacteriophages after multi - pass high - pressure homogenization treatments at 100 mpa in reconstituted skim milk . values correspond to viable phage particles ( pfu / ml ) of ( ) untreated samples , and after ( ) one , ( ) three , and ( ) five passes . similarly to hph , hhp has been proposed as an alternative to the thermal treatments applied in food preservation ( patterson , 2005 ) . advantages of hhp ( preservation of the fresh taste and nutrient content , among others ) in comparison to heat processes are comparable to those previously presented for hph . besides these advantages , effectiveness of hhp in inactivating different microbial species has been reported ( ananta and knorr , 2009 ) . regarding bacteriophage inactivation , scarce data are available , although some authors have documented hhp inactivation of dairy phages . specifically , l. lactis phages p001 and p008 , suspended in enriched m17-broth , were treated at up to 600 mpa ( mller - merbach et al . , 2005 ) . both phages are commonly found in german dairy plants , being representative of c2 ( prolate head ) and 936 ( isometric head ) species , respectively . the isometric phage p008 was considerably more resistant , with a 5 log order reduction after 2 h at 600 mpa , whereas the same titer reduction was obtained for prolate phage p001 during the pressure - build - up time . the greater inactivation observed for the prolate head - phage led the authors think about a correlation between phage morphology and stability , in agreement with the previous hypothesis of moroni et al . according to the latter authors , the long form of the prolate head make it less stable than the isometric structure , thus turning prolate head - phage particle more sensitive to high - pressure treatments . raw milk is regarded as one of the main sources of phages , thus representing their most important way of entry to the industrial environment ( everson , 1991 ; josephsen and neve , 1998 ) . moreover , the capability of bacteriophages to remain in the air for long periods makes bioaerosols one of the most important dispersion routes of virions . unfortunately , none of the currently available technologies is appropriate to inactivate bacteriophages suspended in the air of dairies , either in laboratory or in processing plants ( quiberoni et al . , 1999 , 2004 , 2006 ) . the photocatalytic properties of tio2 have been the subject of much research , mainly for the photochemical pollutant oxidation . several advantages of photocatalysis make this methodology an excellent alternative to the traditional chemical disinfection : ( i ) absence of residues , ( ii ) simultaneous treatment of diverse pollutant mixtures , ( iii ) broad range , and ( iv ) ease of operation . besides , the low cost , high abundance and safety of tio2 make this compound the most frequently selected catalyst ( hoffmann et al . , 1995 ; fujishima et al . , 2000 ; de lasa et al . , 2005 ) . semiconductor tio2 generates highly oxidizing species ( o2-andoh ) when photoexcited by uv radiation , thus catalyzing various chemical reactions , including the decomposition of organic compounds and destruction of microorganisms ( horie et al . , 1996 ) . photocatalysis application has been mostly intended to destroy fungi , bacteria , and spores in the air ( kakita et al . , 2000 ; , 2001 ; koziumi and taya , 2002 ; khn et al . , 2003 ; cho et al . , 2005 ; pal et al . , 2007 ; ditta et al . , 2008 ; zacaras et al . , 2010 ) though its efficiency for the inactivation of viruses contained in bioaerosols has been scarcely explored . furthermore , only a very few studies have been focused on the combined application of uv and tio2 in order to avoid phage dissemination in the environment . sjogren and sierka ( 1994 ) were able to reduce the titer of a ms2 phage ( specific for escherichia coli ) suspension by a factor of 10 after 10 min of irradiation at 2 mw / m in the presence of tio2 in a continuously stirred batch reactor . ( 2000 ) have reported the inactivation of phage pl-1 ( l. casei ) by using a ceramic preparation coated with a mixture of oxides including tio2 and ago . the experiment was conducted in liquid medium and the suspended phages were subjected to irradiation with black - light ( bl ; 300400 nm ) in the presence of tio2 thin film . these authors demonstrated the conversion of the virions into inactivated ghost particles , having an empty head without dna . later , kashige et al . ( 2001 ) investigated the pl-1 phage inactivation mechanism ; the authors found that the genome dna inside the phage capsids was fragmented , suggesting a previous damage to the capsid protein by active oxygen species generated in the tio2 film - settled aqueous medium under bl . the generation of both superoxide anions ( o2- ) and hydroxyl radicals ( oh ) in the aqueous medium was verified by chemical analysis , whereas electrophoresis confirmed a considerable phage genome dna fragmentation . more recently , tio2 photocatalysis was reported as a feasible methodology for the inactivation of phages infecting several lab species , since this technology seemed to be very efficient to this purpose ( briggiler marc et al . , 2009 , 2011 ) . to our knowledge , these studies are the first ones referring photocatalysis ( uv radiation and tio2 ) applied to phages in bioaerosols . ( 2009 , 2011 ) consists of an uv - a radiation emitting system ( wavelength range of 300420 nm , maximum emission at about 350 nm ) , an irradiation chamber , and a support to hold the tio2-coated borosilicate glass plates ( zacaras et al . , 2010 ) . the photocatalytic inactivation was applied on phage suspensions ( 10 plaque forming units ( pfu)/ml ) prepared in distilled water . phage suspensions were taken in the tio2-coated plates , further covered with a borosilicate glass to create a thickness similar to bioaerosol drops , and finally irradiated in a chamber with uv light for a maximum period of 3 h. very dissimilar behaviors were demonstrated by each bacteriophage under the same photocatalytic treatment . pfu / ml ) were achieved for phage bym after 45 min of exposure , but 60 min were necessary to attain similar results ( < 50 pfu / ml ) for phages atcc 8014-b1 and atcc 8014-b2 . likewise , undetectable counts ( < 10 pfu / ml ) of phages j-1 and qf9 particles were reached after 120 min of exposure . contrarily , phages atcc 15807-b1 and ib3 were able to resist up to 180 min of treatment , exhibiting viability losses of only 1.2 log orders . a similar performance was demonstrated by phage mlc - a , with a loss of viable particles of only 2.4 log orders after 120 min of exposure , ( briggiler marc et al . , 2009 , 2011 ) . regarding the photocatalytic inactivation efficiency ( abs ) , values calculated by the authors differed in more than two - log orders among all the phages tested ( table 3 ) . the highest inactivation efficiency was experimented by the bacteriophage atcc 8014-b2 ( abs = 4.244 10 pfu / photon ) , whereas the lowest was that calculated for the phage cb1/342 ( abs = 0.171 10 pfu / photon ) . besides , the authors observed that related phages , lytic for the same strain and morphologically similar , evidenced different behavior under the same conditions of photocatalytic inactivation . attempting to explain these results , a dissimilar stability of viral proteins against the absorbed radiation energy considering the capsomers as a protective shield for viral dna against the damage caused by the photocatalysis , thus different primary and conformational structure of capsid proteins would be relevant factors affecting the resulting inactivation kinetics ( briggiler marc et al . , articles focusing on the photocatalytic inactivation of microorganisms and or viruses hardly report their efficiency in terms of photons absorbed . in this sense , the only known study concerning tio2 photocatalytic inactivation expressed in terms of photons absorbed was applied on bacterial cells , specifically on e. coli suspensions ( marugn et al . , 2008 ) and the kinetic analysis was reported . an apparent photonic efficiency in the order of 10 cfu / photon was calculated by the authors for the maximum e. coli disinfection rate achieved during the experiments . comparable inactivation orders ( of approximately 10 pfu / photon ) were obtained by briggiler marc et al . efficiency of photocatalytic inactivation of dairy bacteriophages ( data adapted from briggiler marc et al . , classification of the international committee on taxonomy of viruses ( ictv ) as illustrated by ackermann ( 2007 ) . briggiler marc et al . ( 2009 , 2011 ) demonstrated the efficiency of the photocatalysis as a new methodology to be applied in dairies , where lab represent the target of bacteriophage attacks . in comparison to the uv - c and uv - c / tio2 units currently available in the market for the environmental air purification ( kover synergy , 2010 ; novapure , 2010 ) , the uv - a radiation assayed by the authors has the advantage of safe use , thus allowing their application for long periods even in the presence of personnel . additional research , particularly focused on the photocatalysis inactivation mechanism and the method efficiency , are indispensable for the design of equipment to be used in industrial environments , either in plants or in laboratories . thermal treatments such as pasteurization and sterilization were traditionally used in food industry for assuring safety and longer shelf - life of food products . although they are efficient and economical processes to inactivate microorganisms , they can cause undesirable protein denaturation , non - enzymatic browning , and loss of vitamins and volatile flavor compounds . bearing in mind the idea of avoiding undesirable consequences of heating , in the last years considerable efforts were focused on the development of novel non - thermal preservation processes . among them , high - pressure processing is one of the most promising , because it combines maximal retention of the chemical and physicochemical product properties with efficient germ reduction ( lado and yousef , 2002 ; diels and michiels , 2006 ) . the most studied and applied pressure - based processes are high hydrostatic pressure ( hhp ) and high - pressure homogenization ( hph ) . although the apparent similarities between both technologies , they have some relevant differences that should be pointed out . first of all , they have very dissimilar exposure time ; this parameter abruptly diminishes from a few minutes in hhp to a second or less in hph ( wuytack et al . , 2002 ) . hph is a high hydrodynamic process during which the fluid is forced to pass through a small orifice and then subjected to an ultra - rapid decompression . the sudden fall in the local pressure of the fluid at constant temperature lead to the nucleation and growth of vapor bubbles ( or cavities ) within the body of liquid , the collapse of which could transmit several localized forces to surfaces or particles , including the microbial cell ( middelberg , 1995 ) . on the other hand , hhp systems consist of a pressure vessel , the pressure transmission fluid ( usually water ) and one or more pressurizing pumps . the packaged food is loaded into the vessel , the top is closed and the fluid is pumped into the vessel from the bottom . when the desired pressure is reached , pumping is stopped , valves are closed and the pressure is maintained without further need for energy input . the high pressure is applied isostatically so that all parts of the food are subjected to the same pressure at exactly the same time . in the case of liquids , such as fruit juices , the whole vessel can be filled with the juice , which becomes itself the pressure transmission fluid ( patterson , 2005 ) . homogenization was developed many years ago with the main purpose of enhancing the texture , taste , flavor , and shelf - life of food emulsions , particularly dairy products like milk and cream butter . as the consumers began to claim for more natural and fresh foods with improved stability , safety and extended shelf - life , a new generation homogenizers was consequently developed , giving as a result the origin of hph ( diels and michiels , 2006 ) . besides the food industry , hph is frequently applied in the pharmaceutical , cosmetic , and chemical industries for the preparation or stabilization of emulsions and suspensions , or to yield viscosity changes in products ( floury et al . , 2004 ) . hph - treated milk seemed to be very convenient to improve cheese quality and physicochemical properties of fermented milks as well ( burns et al . , hph could be also applied to cell disruption of yeasts and bacteria , thus causing a partial inactivation of the microbial population and allowing the release of intracellular components ( guerzoni et al . , 1999 ) . as a consequence , hph has been regarded as an alternative to other processes commonly applied for the inactivation of pathogenic and spoiling microorganisms ( heat , sterilization , and irradiation ) ( diels et al . , 2005 ; diels and michiels , 2006 ) . viral inactivation by hph treatments was not profoundly studied , especially when distinctively speaking of bacteriophages ( moroni et al . , 2002 ; diels and michiels , 2006 ; capra et al . , 2009b ; dsouza et al . , 2009 ) . as it was previously mentioned , raw milk is regarded as the main source of phages entering to the dairies , some of them possessing a remarkable thermal resistance ( capra et al . , 2004 ; briggiler marc et al . , 2009 ) . hph could be therefore used as an unconventional treatment alternative to heat . nevertheless , only a few reports are available in the bibliography . by using electron microscopy , moroni et al . ( 2002 ) observed that hph - treated lactococcal phages were possibly inactivated by the loss of genetic material from fragmented phage heads . either total or partial loss of phage tails was also observed , rendering them incapable of adsorption to the bacterial cell wall . results obtained by the authors demonstrated a statistically significant difference in sensitivity to hph between the two morphology - types of phages : prolate - headed ( phage c2 ) and isometric - headed ( phages sk1 and ul36 ) , being the prolate head less stable than the isometric one . more recently , phages infecting several species of lactic and probiotic bacteria were also studied . amongst them , two temperate lb . paracasei phages ( ilp84 and ilp1308 ) showed a logarithmic reduction of viability along with the successive passes at 100 mpa when suspended in rsm ( mercanti et al . , 2012 ) . for these phages , falls of 4 and 4.5 log orders were observed , respectively , after five passes at 100 mpa ( figure 2 ) . these results were similar to those previously obtained for phages mlc - a ( specific for lb . helveticus ) subjected to the same experimental conditions ( capra et al . , 2009b ) , observing viability losses of 3.8 , 3.75 , and 2.0 log orders , respectively . in turn , phages mlc - a8 ( lb . casei ) , bym ( lb . delbrueckii ) , alq13.2 ( s. thermophilus ) , qp4 and qf12 ( l. lactis ) were not detected after only five passes at 100 mpa through the high - pressure homogenizer . it is interesting to emphasize that related phages mlc - a and mlc - a8 , lytic for the same probiotic lb . paracasei strain , showed remarkably different behavior after hph treatment , being the former more resistant than the latter . in addition , hph treatments applied to different initial concentrations of the same phage rendered dissimilar inactivation efficiency . contrarily to earlier reported results ( moroni et al . , 2002 ; capra et al . , 2009b ) , working specifically with phage mlc - a , found that the higher the initial phage load , the greater the reduction achieved . on the other hand , bacteriophage inactivation rate showed to be proportional to both applied pressure and number of passes . concerning the influence of suspension media ( milk , whey permeate , buffer ) , results were very dependent on each phage . although some authors ( moroni et al . , 2002 ) reported diminished hph effectiveness in milk and whey permeate , no clear protective effect for any of the assayed media was detected by capra et al . viability of dairy bacteriophages after multi - pass high - pressure homogenization treatments at 100 mpa in reconstituted skim milk . values correspond to viable phage particles ( pfu / ml ) of ( ) untreated samples , and after ( ) one , ( ) three , and ( ) five passes . similarly to hph , hhp has been proposed as an alternative to the thermal treatments applied in food preservation ( patterson , 2005 ) . advantages of hhp ( preservation of the fresh taste and nutrient content , among others ) in comparison to heat processes are comparable to those previously presented for hph . besides these advantages , effectiveness of hhp in inactivating different microbial species has been reported ( ananta and knorr , 2009 ) . regarding bacteriophage inactivation , scarce data are available , although some authors have documented hhp inactivation of dairy phages . specifically , l. lactis phages p001 and p008 , suspended in enriched m17-broth , were treated at up to 600 mpa ( mller - merbach et al . , 2005 ) . both phages are commonly found in german dairy plants , being representative of c2 ( prolate head ) and 936 ( isometric head ) species , respectively . the isometric phage p008 was considerably more resistant , with a 5 log order reduction after 2 h at 600 mpa , whereas the same titer reduction was obtained for prolate phage p001 during the pressure - build - up time . the greater inactivation observed for the prolate head - phage led the authors think about a correlation between phage morphology and stability , in agreement with the previous hypothesis of moroni et al . according to the latter authors , the long form of the prolate head make it less stable than the isometric structure , thus turning prolate head - phage particle more sensitive to high - pressure treatments . phage control measures should be focused on avoiding dissemination of phages , so as to maintain their titers under critical levels . diverse chemical and physical treatments industrially employed for sanitization showed different rates of efficiency on phage inactivation . considering thermal treatments , either ltlt or htst pasteurization conditions would not assure a complete inactivation of phage particles , especially of those very thermo - resistant , and even 90c were not enough to inactivate some dairy phages . among biocides , only peracetic acid remains the only fully reliable option for phage destruction , though two new products seem promising for this purpose . the former attained optimistic results until now and appears to be suitable for diminishing viable phages suspended in the air but , however , further research is needed . high - pressure treatments were barely tested for phage inactivation as well ; they would fairly preserve cheese - making aptitude of milk , in contrast to strong thermal treatments , but tested phages exhibited in general a high resistance to high pressure . anyhow , two or more inactivating strategies ( maybe combining non - thermal with thermal processes ) should be applied either in parallel or consecutively to obtain better results . future efforts should be directed to determine the best combinations which permit generating a synergistic effect and thus softening the strength of each individual treatment while accomplishing the highest phage - killing activity possible . 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 .

the greatest burden of diarrhea is in children under 5 years of age in developing areas . in fact , childhood diarrhea accounts for almost one million directly attributable deaths per year globally . additionally , morbidity associated with repeated episodes of childhood diarrhea can be long - lasting . collectively , the diarrheagenic escherichia coli ( dec ) represent the most common bacterial pathogens worldwide . some of the dec pathotypes represent a major cause of morbidity and mortality in developing and low - income countries ; these pathotypes include the enteropathogenic e. coli ( epec ) , enterotoxogenic e. coli ( etec ) , enteroinvasive e. coli ( eiec ) and enteroaggregative e. coli ( eaec ) . in contrast , the shiga toxin ( stx)-producing e. coli ( stec ) , a dec which can cause bloody diarrhea , constitute a challenge for public health systems in industrialized countries . the epidemiology of infections caused by dec ranges from sporadic endemic diarrhea to small or large outbreaks of food- or water - borne diarrhea . some of the most severe and well - known dec outbreaks have been due to stec , particularly the o157:h7 serotype . o157:h7 , referred to by some authors as enterohemorrhagic e. coli ( ehec ) , are known to cause sporadic cases and outbreaks of hemorrhagic colitis and a potentially lethal sequelae : hemolytic uremic syndrome ( hus ) . hus is defined clinically by the triad of hemolytic anemia , thrombocytopenia , and renal failure . hus is the most frequent cause of renal failure in children , and shiga toxin is the critical stec virulence factor associated with the development of hus . shiga toxins are classified as belonging to one of 2 types , stx1 or stx2 , and within the 2 types there exist several subtypes . however , o157:h7 e. coli strains that produce stx2a are more likely to be associated with hus . from may through june 2011 , an unprecedented outbreak of gastroenteritis occurred in germany , with 4,137 known affected individuals and 54 deaths . in addition to the stx2a - encoding phage , the o104:h4 isolate carried the extended - spectrum -lactamase ( esbl ) antibiotic resistance plasmid . the consumption of fenugreek - sprouted seeds imported from egypt was identified as the most likely source of infection for primary outbreak cases . implication of o104:h4 in a massive outbreak was remarkable , but equally remarkable was the high proportion of cases developing hus : 22% of the recognized o104:h4 cases were diagnosed with this complication contemporaneously . in contrast , historical rates of hus after o157:h7 infection typically range from 6%-15% . although passive clinical surveillance can not accurately ascertain the rate of any complication , it is indisputable that the outbreak strain was unusually virulent for a non - o157:h7 strain . interestingly , whereas the o104:h4 strain carried a stx2a phage characteristic of hus associated stec strains , it did not possess other o157:h7 virulence traits , most notably the locus of enterocyte effacement ( lee ) pathogenicity island , which promotes the distinctive attaching and effacing epithelial lesion associated with lee positive stec . instead , the o104:h4 outbreak isolate harbored genes characteristic of a completely different dec pathotype , eaec . such an unusual combination of stx with virulence factors typically found in eaec has been documented prior to the 2011 german outbreak . this include reports of sporadic small outbreaks of hus attributed to stx - producing eaec of the o111:h2 , o111:h21 and , yes , o104:h4 serotypes . nevertheless , these cases or outbreaks were localized to small populations in france , ireland , and the republic of georgia and were not spread throughout europe , as seen with the german outbreak strain . we will argue that they are not in fact hybrid strains , but are rather eaec that have been infected with stx - encoding phage and should be designated stx - eaec . eaec is a cause of acute and persistent diarrhea in multiple settings and eaec strains express a heterogeneous array of putative virulence factors encoded on the bacterial chromosome or on the eaec - specific paa plasmid . eaec strains often harbor a transcriptional activator of the arac / xyls class called aggr , which controls expression of genes on both the plasmid and the chromosome . genes under aggr control include those that encode the aggregative adherence fimbriae ( aaf ) , of which at least 5 variants exist ( aaf / i - v ) . aaf adhesins are essential for eaec adherence to human intestinal explants , and for both cytokine release and opening of epithelial tight junctions in a polarized epithelial model . aggr is also required for expression of genes encoding dispersin ( the aap gene ) , the aat dispersin translocator and a chromosomal cluster termed aai , encoding a type vi secretion system . described a novel eaec regulator called aar ( aggr - activated regulator ) , which is a member of a previously unrecognized large class of regulators in pathogenic gram negative bacteria . the identified aar gene is activated by aggr , however when aar is deleted , aggr and the aggr regulon remain persistently activated . thus , aar could act directly or indirectly as a virulence suppressor as it down - regulates the expression of the positive regulator aggr . however , aar is present in the german o104:h4 outbreak strain , so the exact contribution of aar to eaec pathogenesis remains enigmatic . additionally , eaec strains also often harbor a variable number of serine protease autotransporters of enterobacteriaceae ( spates ) that are implicated in immune evasion , mucosal damage , secretogenicity , and colonization . limited knowledge exists on eaec of the o104:h4 serotype . in a recent study of children 's diarrhea in mali , we identified stx - negative eaec o104:h4 in 6 children with and without diarrhea . whole - genome sequencing of the stx2a+ o104:h4 outbreak strain , stx - negative o104:h4 malian eaec isolates and other o104:h4 isolates confirmed that the outbreak strain was indeed genetically eaec . genomic forensics suggested that the o104:h4 outbreak strain was an eaec that had acquired the stx2a phage because the outbreak strain was phylogenetically clustered with typical eaec of o104:h4 and other serotypes . in addition , the genome analysis demonstrated that , other than stx2a , the outbreak strain contained no virulence factors not already described in eaec strains , including the aaf variant i ( aaf / i ) , aap / dispersin , the aat translocator , the aai type vi secretion system , the aggr regulator , and 3 spate proteases ( pic , siga and sepa ) . that said , spate proteases are present in pathogenic e. coli strains and shigella spp ; however , the confluence of pic , siga , and sepa is a combination typically found among shigella flexneri 2a strains , a finding that suggests that the german outbreak strain shares characteristics with that highly invasive and inflammatory diarrheagenic pathogen . so why was the o104:h4 eaec outbreak strain so virulent ? and which genes or combinations of genes could explain the high hus incidence rate in patients ? answering these questions is challenging , given that eaec are human - specific pathogens and that few animal infection models mimic human disease . however , zangari et al . recently investigated the virulence of the german o104:h4 outbreak strain in a mouse model and showed that stx2a from the outbreak strain was associated with weight loss , renal impairment , and death . by contrast , those factors associated specifically with typical eaec virulence are less clear because eaec is a highly heterogeneous group of pathogens . eaec as currently defined most likely encompasses both pathogenic and non - pathogenic e. coli strains . nonetheless , eaec has unmistakably been associated with diarrhea in some individuals ( e.g. in volunteers and outbreak patients ) , subclinical colonization in endemic areas is common , urinary tract infections and sepsis , and recently with poor growth in children . the essential differences between pathogenic and non - pathogenic strains are mostly unknown , but pathogenesis both ex vivo and in vivo suggest that the current pathogenic scheme of eaec is comprised of these 3 stages : ( 1 ) after ingestion and passage through the stomach , eaec adheres to the intestinal mucosa within a mucus - containing biofilm by virtue of aaf and possibly other adherence factors such as pic mucinase and dispersin ; ( 2 ) stimulation of mucus production following biofilm formation at the surface of the mucosa ; and ( 3 ) inflammation of the mucosa , manifested by cytokine release , cell exfoliation , and intestinal secretion . the final pathogenic step described previously is mediated as follows : both aaf and flagellin induce an inflammatory response in intestinal epithelial cells , manifested by increased cytokine production , as well as fecal lactoferrin in stools of patients with eaec - induced diarrhea . spate toxins ( such as pet , siga , sat ) cause cytotoxicity due to alteration of cytoskeletal elements . based on the above outline of the stages of eaec - mediated gastroenteritis , we hypothesized that the plasmid - borne virulence factors of eaec contributed to the high pathogenicity of the german outbreak strain by promoting strong adherence to the epithelium and/or by opening epithelial tight junctions and facilitating stx2a translocation . we used isolate c227 - 11 from the german outbreak , and investigated the potential contribution of paa with mutants of c227 - 11 , either cured of the paa plasmid or deleted individually for known paa - encoded virulence - associated genes aggr , agga , or sepa . as expected , lack of the paa plasmid abolished the capacity of c227 - 11 to adhere to viable colonic tissue harvested from the cynomolous monkey macaca fascicularis . in contrast , c227 - 11 adheres in an aggregative manner and forms heavy biofilms with a thick mucus layer upon interaction with the monkey colonic tissue ( fig . adherence is followed by mucosal toxicity , including crypt dilation , microvillous vesiculation , and epithelial cell extrusion . we also investigated the role of aaf / i in biofilm formation and epithelial cell adherence for c227 - 11 , and found that like variants aaf / ii , iv and v , expression of the agga fimbrial adhesin was necessary and sufficient for adherence and biofilm formation ( not shown ) . 2 shows an electron micrograph of c227 - 11 in which aaf / i can be visualized . sections of proximal colon from freshly euthanized cynomolgus monkeys were incubated with strain c227 - 11 ( a ) , with hs ( b ) or with media alone for 3 hours ( c ) and then visualized by scanning electron microscopy ( sem ) . in panel ( a ) , the arrow indicates dilation of the crypts . . figure 2.scanning electron microscopy of c227 - 11 and biofilm formation after 18 hours on glass cover slips . abbreviations : aaf / i ; aggregative adherence fimbriae type i , paa ; virulence plasmid of c227 - 11 , esbl ; extended - spectrum -lactamase antibiotic resistance plasmid , aggr ; arac / xyls family activator , aai ; type vi secretion system , aat ; dispersin translocator , stx ; shiga toxin , siga ; iga protease - like homolog , pic ; serine protease precursor , and sepa ; shigella extracellular protease . sections of proximal colon from freshly euthanized cynomolgus monkeys were incubated with strain c227 - 11 ( a ) , with hs ( b ) or with media alone for 3 hours ( c ) and then visualized by scanning electron microscopy ( sem ) . in panel ( a ) , the arrow indicates dilation of the crypts . scanning electron microscopy of c227 - 11 and biofilm formation after 18 hours on glass cover slips . abbreviations : aaf / i ; aggregative adherence fimbriae type i , paa ; virulence plasmid of c227 - 11 , esbl ; extended - spectrum -lactamase antibiotic resistance plasmid , aggr ; arac / xyls family activator , aai ; type vi secretion system , aat ; dispersin translocator , stx ; shiga toxin , siga ; iga protease - like homolog , pic ; serine protease precursor , and sepa ; shigella extracellular protease . we next interrogated cytoskeletal rearrangement in cells infected with c227 - 11 , because such rearrangement has been demonstrated for other eaec and has been linked to the presence of aaf fimbriae . in addition , disruption of the epithelial barrier coincides with perturbation of the actin cytoskeleton for stec strains that carry the lee island . however , as mentioned previously , c227 - 11 does not harbor the lee island . we found that disruption of the actin cytoskeleton and reductions in transepithelial resistance in t84 cells infected with c227 - 11 were dependent on aggr and agga but not on the stx2a phage . thus , the contribution of aggr in altering actin arrangement ( and reducing adherence ) is likely due to its role in the expression of aaf / i . we also found that a prototype o157:h7 failed to decrease transepithelial resistance to the same extent as eaec , supporting the notion that it is aaf or aaf - dependent factors rather than stx2a that are responsible for epithelial barrier loss in this model . these observations were confirmed by expressing the aaf / i fimbrial adhesion in an e. coli k12 background , which proved sufficient to enhance translocation of stx2a across the epithelial monolayer . stec strains associated with hus possess several t3ss - associated effector proteins that can disrupt the integrity of the epithelial junctions and possibly contribute to stx penetration . however , we suggest that aaf - associated tight junction disruption caused by the german outbreak strain more plausibly explains the increased rate of stx - related complications induced by this strain . we also observed that incubation of c227 - 11 with infected cell monolayers released the chemokine interleukin 8 ( il-8 ) , and that cytokine release was significantly lower when the cells were infected with the strain cured of the stx2a phage ( c227 - 11cu ) . in addition , deletion of agga , aggr , or sepa significantly decreased secretion of il-8 into the basolateral compartment . furthermore , when expressed in k12 , the agg - fimbrial cluster from c227 - 11was sufficient to induce a pro - inflammatory response , as previously reported for the eaec aaf / i prototype strain , jm221 . taken together these results suggest that induction of an inflammatory response by c227 - 11 is multifactorial and depends on both stx2a and eaec factors . we suggest a model for the pathogenesis of the o104:h4 outbreak strain that include steps listed above for eaec alone , but incorporates additional features explained by the expression of stx2a ( fig . 3 ) . ( 1 ) o104:h4 adheres to the intestinal mucosa within a mucus - containing biofilm , by virtue of aaf and possibly other adherence promoting factors such as the pic mucinase and dispersin . ( 2 ) interactions between aaf / i and the epithelial monolayer causes disruption of the epithelial barrier and actin cytoskeletal rearrangement . ( 3 ) aaf / i , sepa and stx2a triggers release of inflammatory cytokines and recruitment of neutrophils . ( 4 ) siga causes cytotoxicity possibly due to alteration of cytoskeletal elements . ( 5 ) aaf / i - mediated disruption of the epithelial barrier in addition to para - cellular passage of neutrophils facilitates the delivery of stx2a to the lamina propria , with increased access to intestinal vasculature and , ultimately , to the target organs that express high levels of the stx2a globotriaosylceramide receptor , gb3 ( particularly , kidneys and brain ) . our findings thus suggest a mechanism whereby the o104:h4 outbreak was associated with unusually high rates of hus . our studies demonstrate the importance of the paa plasmid in the pathogenesis of the o104:h4 outbreak strain , both in ways typical of eaec ( adherence and inflammation ) , and in a unique and sinister new synergy ( barrier dysfunction that augments stx2a translocation ) . consistent with our results is the observation that a proportion of o104:h4 isolates during the outbreak had lost the paa plasmid , and patients whose initial isolate expressed the plasmid were significantly more likely to develop hus than those infected with a paa - negative strain . given the virulence of the stx - eaec o104:h4 strain it would be relevant for the health community to consider , if the acquisition of the stx2 phage by an eaec is an uncommon event or something likely to occur more frequently . as mentioned , the german outbreak was not the first clinically linked instance of a stx - eaec , but it is an example of such a strain causing a large- scale outbreak of disease . however , whether the german outbreak was due to a particularly virulent stx - eaec , a rare epidemiologic opportunity , or both is unclear . concluding , the emerging of stx - eaec strains possesses a major challenge for the public health , which needs to implement diagnostic procedures that identify the most virulent clones and this is particular challenging with this heterogeneous pathotype . the work described here was supported by ai-033096 to jpn , r37 ai020148 and r0731977 to ado and dff1333 - 00156 to nbo .

it was the time when an ambitious early career researcher finished writing the manuscript of his master s thesis . he tried to sort through a number of journals , though he was not sure which journal was the right one to publish his work . email from a journal pledging publication within a month . that was the time this young scientist became the victim of a predatory journal . publishing is an essential step to reach a wider audience and it is a key metric to measure the maturity of scientists ( 1 ) . however , for many young academicians , particularly from developing countries , the question of where to publish their work is a considerable challenge . submitting a manuscript to an inappropriate journal is one of the common mistakes of early career scientists ( 1 ) . in developing countries , where institutions and libraries can not afford the subscription fees to access journal articles , open access is the best model to reach those who can not afford to access relevant scientific publications ( 234 ) . motivated by press freedom , global internet and financial gains , predatory journals have emerged to corrupt open access . everyone working in science daily receives emails of article submission invitations from journals and publishers . they request manuscript processing fees which mainly guarantees the acceptance of manuscripts without adequate scientific review ( 56 ) . they request payments from those who can not even afford to publish manuscripts ( 57 ) . predatory journals and publishers are those having minimal or nonexistent peer review , allowing weak scientific content to be published in the name of authentic science ( 5 ) ( fig . the 2016 jeffrey beall list shows that 923 publishers , 882 standalone journals and 101 hijacked journals are registered as predatory ( 8) . inexperienced researchers from developing countries are the victims of these junk journals ( 9 ) . promotions of academic ranks in developing country universities are based on the number of publications rather than quality ( 1011 ) . in 2014 , more than three quarter of the authors were from asia and africa ( 12 ) . these journals are polluting the academic world as university positions are getting to be filled with people having poor quality publication profiles ( 11 ) . they received 10 - 15 journal article submission and conference invitations in their inbox and spam folders from predatory publishers every day . for 28 article submission invitations , one of the victims replied : dear predatory publishers , please stop writing article submission invitation emails . i will send your journal to jeffrey beall so that your journal will be added in the predatory journals list . one of the so called editors in chief replied with broken english : well , sir , you do not be angry , i am your email removed but do not journal no fraud occurred . in many occasions , young authors bargain with the publishers to reduce the requested publication fee . others described that they pay the requested publication fee through third party payers such as through their relatives living abroad . authors from low - income countries need to be aware that they should not pay for publishing . one junior scientist declared that being the victims of these junk publishers has made me feel the pain at least after a year , and after receiving so many rubbish article submission invitations through emails . i asked myself , are these journals really good journals , and is it possible to identify good journals from a fake one ? that was the time i taught myself about these counterfeit journals . the more collaborative a research is , the higher the quality it is and the more likely it will be submitted to a reputable journal . senior faculty members of developing country universities need to coach young scientists on publication processes . the often used measure of identifying the credibility of journals is thomson and reuters impact factor ( 14 ) . the name thomson and reuters impact factor which authors shortly know as impact factor of journals is hijacked and the fake measure called journal impact factor has emerged . beall has listed more than 50 misleading metrics and questionable companies providing these metrics ( 8) . authors need to make sure that whether the metrics provided by the journal are from recognized bodies such as thomson and reuters or scimago journal and country ranks . the problem is increased when these dubious articles published in predatory journals are cited as references in good quality peer reviewed journal articles which in turn lead for poor quality research outputs to have citation reports . hence , credible journals and peer reviewers need to prevent citation by requesting authors to remove references published in predatory journals . one of the victims of these journals has 12 publications and half of them are on the jeffrey beall s list of predatory journals . there are a number of other inexperienced researchers who achieved research positions , assistant professor as well as in the associate professorship positions , by accumulating poor publication accounts . the frustration to be promoted is one of the key reasons why emerging scientists from developing countries are knowingly or unknowingly submitting their manuscripts to the predatory journals . to solve this problem , university councils responsible for promoting academic staffs to higher academic positions need to have a regulatory framework or revise their senate legislations for preventing the promotion requests of researchers having poor publication accounts . publications should be carefully checked before approving the promotions and articles from predatory journals should not be counted . a senior faculty member in one of the universities in ethiopia searched his name in google scholar and found a new publication which he did not approve the manuscript before submission . in fact , the article was published in one of the fast - track predatory journals . supervisors of the students need to make sure that the article shall not be submitted unless they have approved the submission . they have to follow and coach their students on manuscript preparation and publishing in legitimate journals . it is ambitious to claim that the global scientific community should come together and appeal for firm actions to be taken against this misconduct . closing down the web sites of these fake journals and publishers or suing them for their alleged cybercrime is unconvincing . instead , we have to alert our students , and the scientific community around the world , especially in developing countries . a recent study reported awareness campaigns and consultations with information facilitators could make a difference in tackling the predatory publishing practice ( 15 ) . course contents of the undergraduate and postgraduate curriculums need to include mechanisms of checking the authenticity of a journal , credibility of the metrics and whether the journal is indexed in reputable databases such as medline , pubmed or web of science . we recommend authors mainly not to rely on pubmed to check the reputability of a biomedical journal . authors should also check whether the journal is from a credible publisher , affiliated research institute or university , has a transparent peer review process and availability of the journal contact details ( especially postal address and telephone number ) . moreover , authors may consider to consult journal / author name estimator ( jane ) website ( http://jane.biosemantics.org/ ) to look for potential reputable journals for their particular topic ( 16 ) credible journals do not chase authors and send article submission invitations , rather authors look for them . however , legitimate publishers and journals also use email to reach potential authors on a regular basis . we have contacted jeffrey beall in case he would like to have the spam emails forwarded to him . i am always happy to have people forward me the spam emails they receive when they believe they have discovered a new questionable publisher or journal . he also stressed : before forwarding these emails , authors should first check whether the name of the publisher or a journal is in the beall s list . frequently , predatory publishers release new journals . hence , authors should check whether these journals are included in the beall s list of predatory publishers . if the publisher is already in the list , there is no need to forward these article submission invitations . the emergence of new predatory publishers , fake journals , and their fake metrics , fake conferences and publishing in the predatory journals is likely to continue . an organized database to help authors quickly search and identify whether a journal is predatory needs to be developed . moreover , a concerted multi - country level initiative composed of all stakeholders ( publishers , credible journal editors , reviewers , and researchers ) is required to alleviate the problem and build the legacy that beall has already established and fight the problem on a global scale .

total knee arthroplasty ( tka ) is widely accepted treatment for moderate or severe osteoarthritis and rheumatoid arthritis . significant blood loss can be seen during the early postoperative period where a blood transfusion may be necessary . however , it has been known that the donor blood may lead to an immunological reaction and the transmission of viral infections ( e.g. , hepatitis and aids).1 various methods have been suggested to reduce blood loss following tka , such as preoperative erythropoietin and iron supplementation , autotransfusion,23 postoperative blood salvage,4 hypotensive anesthesia,5 tranexamic acid administration,6 intraarticular epinephrine injection,7 thrombin based hemostatic agent,8 and temporary drain clamping.910 closed suction drainage is known to prevent the formation of hematomas in the operative field , decrease tension on incisions , diminish delayed wound healing and reduce the risk of infection.11 however , some studies have demonstrated that closed drainage leads to increased blood loss after tka because it eliminates the tamponade effect and may cause a retrograde infection.12 the role of wound drainage following knee arthroplasty is still controversial.1314 despite these disagreements , wound drainage is a procedure widely used by orthopedic surgeons and it is still assumed to be a standard of care and suggested for use in tka . subcutaneous indwelling closed suction drainage method has been known to be beneficial and an alternative to the intraarticular indwelling method.15 this method provides tamponade effect as the joint is not drained and in addition has subcutaneously drainage effect preventing the soft tissue hematoma and wound problems . the purpose of this prospective randomized study was to compare the visible , hidden , total blood loss and postoperative hemodynamic change of subcutaneous and intraarticular indwelling closed suction drainage method after tka . the hypothesis was that subcutaneous indwelling method would decrease the visible , total blood loss and reduction of postoperative hemoglobin ( hb ) or hematocrit ( hct ) change after tka . 160 patients with primary osteoarthritis , who underwent unilateral tka in our institute between january 2011 and september 2011 , were enrolled in this trial . all patients were randomly assigned into two groups ( 80 per group ) using computer - generated numbers . the sample size was calculated to detect a significant difference in the postoperative drainage and laboratory change between groups with a power of 80% with an value of 0.05 . group a and b were patients with subcutaneous and intraarticular indwelling closed suction drainage method , respectively . this left 157 tkas ( 78 patients for group a and 79 patients for group b ) who form the basis of the current report [ figure 1 ] . exclusion criteria include other diagnosis than primary osteoarthritis , preoperative valgus alignment , received a revision or bilateral tka , hematological disease , previous open knee surgery or vascular surgery . there were 53 males and 104 females with an average age of 69.5 5.8 years ( range 61 - 82 years ) , and the average body mass index was 24.9 4.7 kg / m [ table 1 ] . this study was conducted after the approval of the institutional review board and informed consent was obtained from each patient before enrollment in the study . flow char showing consolidated standards of reporting trials patient demographics , preoperative deformities and functional status all surgeries were performed by a single surgeon ( jhy ) using the navigation system ( orthopilot , version 4.0 ; b. braun aesculap , tuttlingen , germany ) under spinal anesthesia . all 160 knees underwent the same surgical approach consisting of a midline skin incision and a medial parapatellar approach under tourniquet . all patients were implanted with cemented type ultra - congruent fixed bearing design ( columbus uc , b. braun aesculap , tuttlingen , germany ) . none of the patients underwent lateral retinacular release or patellar resurfacing . in the subcutaneous indwelling group ( group a ) , a vacuum drainage system was placed in the subcutaneous space ( below the medial skin flap ) after joint capsule closure [ figure 2].15 in the intraarticular indwelling group ( group b ) , a vacuum drainage system was placed in the intraarticular space ( the medial gutter ) before joint capsule closure . the amount of drained visible blood was recorded at 24 and 48 h using the measuring cylinder with total 500 ml and 1.0 ml unit . using gross 's formula,16 hidden blood loss was calculated using patients height , weight , and preoperative and postoperative hct . the hb and hct levels were determined preoperatively and 12 h ( day 1 ) , 5 and 10 day postoperatively . the patients received packed red blood cells if their hb levels decreased to < 8 g / dl and presents compromised clinical criteria ( e.g. , tachycardia , hypotension , or symptoms of anemia that were relative to the preoperative medical condition of the patient ) necessitated transfusion . the postoperative timing of transfusion and the number of units of transfused red blood cell concentrates were recorded . all patients were given low molecular weight heparin ( enoxaparin 40 mg ) subcutaneously daily for 7 days postoperatively . note that the indwelling drains ( white arrows ) are placed subcutaneously superficial to the joint capsule closure after the operation , patients of both groups were encouraged to perform a mechanical ankle pumping exercise to prevent deep vein thrombosis as soon as possible after surgery . the bandage and foley 's catheter were removed on the second postoperative day . on the same day , the range motion ( rom ) exercise using continuous passive motion devices , an isometric / isotonic quadriceps exercise , a straight leg rising exercise and a walking exercise were initiated under the control of a physiotherapist . all the observations were recorded by clinical research orthopedic nurse who was unaware about the allotted groups . oozing persisting beyond 2 days after surgery , subcutaneous hematoma ( requiring aspiration or surgical drainage ) , hemarthrosis ( requiring aspiration or surgical drainage ) , ecchymosis ( larger than 3 cm in diameter ) and wound infection ( requiring additional treatments such as antibiotics coverage or surgical debridement ) were recorded . thigh and calf circumference were checked at day 7 after tka . the level for thigh circumference was set at 10 cm proximal from the patella upper pole and largest diameter for calf . symptom severity was assessed at 24 months using the knee society score ( kss)17 and western ontario and mcmaster universities osteoarthritis index ( womac ) score.18 passive maximum knee rom was measured using a goniometer . at these evaluations , a kss of 90 points was considered an excellent outcome , a score between 80 and 89 points was considered a good outcome , a score between 70 and 79 points was considered a fair outcome and a score of < 70 points was considered a poor outcome . the womac system involves the completion of a 24-item questionnaire with three sections : namely ; pain , stiffness and function.18 five response options are possible ( none , mild , moderate , severe and extreme ) , which are scored from 0 to 4 to yield subtotal scores for pain ( 5-item ; possible total score range 020 ) , stiffness ( 2-item ; possible score range 08 ) , and function ( 17-item ; possible score range 068 ) . the two study groups were compared with respect to blood volumes collected via indwelled drains , changes in hb / hct levels postoperatively , requirements for allogeneic blood transfusion , bleeding - related wound problems , and postoperative hospital stay . the kolmogorov fisher 's exact test or the yates chi - square test was used to analyze categorical variables . the complication rate between the two groups was determined by the chi - square test with fisher 's exact test . the statistical package for social science version 10.1 ( spss inc . , chicago , il , usa ) was used for all analysis and p < 0.05 was considered statistically significant . there were no significant differences between the subcutaneous drainage group ( group a ) and intraarticular drainage group ( group b ) regarding the preoperative demographs [ table 1 ] . the total blood loss after tka was significantly different between two groups ( p = 0.04 ) . the total blood loss ( visible drained blood + hidden loss ) was less in subcutaneous drainage group . the mean visible blood drainage in subcutaneous drainage group was significantly less than intraarticular drainage group , both during the first 24 h and between 24 and 48 h ( p < 0.001 ) . the hidden blood loss after tka was significantly different between two groups ( p < 0.001 ) [ table 2 ] . comparison of hematologic data there were no significant differences in hb or hct level in 2 groups before the surgery . however , significant differences of change of hb were demonstrated between groups at 12 h ( day 1 ) , 5 and 10 day postoperatively [ table 2 ] . allogeneic transfusion requirements between patients in the subcutaneous drainage and intraarticular drainage groups ( 6.4% versus 24.1% ) were significantly different ( p = 0.002 , table 3 ) . comparison of transfusion rate and the units in terms of wound problems [ table 4 ] , none of the patients developed delayed wound healing with skin edge necrosis , wound infection , deep vein thrombosis in this study . subcutaneous drainage group ( group a ) demonstrated a higher rate of minor complications such as the bullae formation and ecchymosis compared to intraarticular drainage group . mean postoperative hospital stay in group a and group b was 13.8 and 14.1 days , respectively ( p > 0.05 ) . comparison of bleeding - related wound problems , change of thigh - calf circumferences and postoperative hospital stay there were no significant differences between groups in functional outcomes at 2 year after surgery [ table 5 ] . the most important finding of this study was that subcutaneous indwelling closed suction drainage method reduces both the visible blood loss and total blood loss ( hemovac drainage + internal blood loss ) thus decreases the rate of allogeneic transfusion . although the incidence of minor complications such as the bullae formation and the ecchymosis were significantly higher in the subcutaneous indwelling group , the functional outcome at postoperative 2 years did not demonstrate the difference from intraarticular drainage group . a few studies have shown that closed negative pressure drainage is beneficial to early knee function recovery by reducing postoperative hematoma formation in the wound , relieving wound tension , preventing complications including pain and poor healing and lowering the incidence of deep infections.19 however , a number of other studies have not supported the advantages of closed negative pressure drainage and indicated that closed negative pressure drainage causes complications such as postoperative hemorrhage , increased blood transfusion and infection and affects the ability of postoperative functional training.122021 the use of vacuum drainage in tka to manage bleeding continues to be debated . because most of the blood loss in tka occurs during the first few postoperative hours ( 37% in 2 h and 55% in 4 h ) , it seems reasonable to clamp the drain tube in the first few hours after tka to temporarily create a tamponade effect for bleeding control . recommendations included no clamping,22 clamping for 1 h,23 10-min clamp releases every 2 h,24 and clamping for 4 h.10 the effect of clamping method is also in controversy . the method of subcutaneous indwelling closed suction drainage had been proposed as a potentially efficacious alternative to either intraarticular closed suction drainage or no drainage system.15 this method was suggested to exploit the advantages of both methods ; the subcutaneous blood drainage and the joint tamponade effect . seo et al.15 have shown that subcutaneous closed suction drainage involves equivalent blood loss with comparable wound problems and functional outcomes compared with intraarticular closed suction drainage . however , only exogenous blood loss was analyzed in their study while internal blood loss ( i.e. , total blood loss ) was calculated in this study . in addition , detailed postoperative minor complications were identified using the parameters such as the change of thigh and calf circumference . the occurrence of bullae formation and ecchymosis were significantly higher in subcutaneous indwelling closed suction drainage group in this series . although some studies have demonstrated no statistically significant difference in hematoma formation with or without postoperative drainage , tka patients without drainage system theoretically have a higher chance of hematoma formation . while pressure dressing reduces superficial bleeding at the incision , it is less effective on bleeding in deep locations such as the marrow and periprosthesis space , which can result in invisible blood loss and hematoma formation due to blood accumulation.25 after tka , because the capsule of the knee joint is damaged and the surrounding soft tissue has been incised , the blood infiltration can extend to the fascia and intramuscular space of the thigh and calf , which results in increase in thigh calf circumference and ecchymosis . subcutaneous indwelling closed suction drainage method may not be a perfect solution to prevent soft tissue accumulation from excessive blood infiltration . despite significantly increased minor wound problems and increased thigh - calf circumference compared to intraarticular drainage group , two groups had similar functional outcome at 2 years postoperatively . soft tissue swelling around the knee joint in subcutaneous indwelling closed suction drainage group might have reduced the rom shortly after the operation , but it did not influence the motion arc in the long run . limitation of this study are methods used regarding the drainage system in tka , subcutaneous indwelling closed suction method was compared with only the intraarticular indwelling drainage method . a group without closed suction drainage system or groups of various clamping methods have not been compared . therefore , conclusions can not be made whether subcutaneous indwelling closed suction drainage method has advantages or disadvantages from other methods . subcutaneous indwelling closed suction drainage method in tka reduces total blood loss and thus lowers the rate of allogeneic blood transfusion compared to the intraarticular indwelling drainage method . although the incidence of minor wound problems such as bullae formation and ecchymosis is higher than the intraarticular indwelling drainage method , eventual motion arc and functional outcome are similar . based on these results ,

the terms " migrated " and " sequestrated " disc or fragment refer to displacement of disc material away from the opening in the annulus through which it has been extruded.5 ) when the distinction between the outer annulus and the posterior longitudinal ligament ( pll ) is unclear and the torn annulus opening is small in sequestrated lumbar disc herniation , optimal surgical treatment is still debatable . williams13 ) reported successful results for a microsurgical approach to a virgin herniated lumbar disc that involved removal of only the free fragment compressing the nerve root . by preserving healthy intervertebral disc material , this procedure supposedly offers excellent success rates , few complications , and a low recurrence rate . however , rogers8 ) reported a high recurrence frequency after surgery involving removal of only the offending fragment as compared with more extensive discectomy . of the therapeutic minimally invasive treatment options currently available , nucleoplasty is commonly performed to decompress nucleus pulposus and lower intradiscal pressure by inserting devices into the affected intervertebral disc.9 ) the purpose of this study was to determine the surgical outcome of microscopic fragmentectomy and intraoperative nucleoplasty in cases of sequestrated lumbar disc herniation . twenty - four patients with magnetic resonance imaging confirmed sequestrated lumbar disc herniation that underwent combined microscopic fragmentectomy and intraoperative nucleoplasty between 2009 and 2012 were included in this study . after sufficient consenting to the surgical procedure , all underwent combined microscopic fragmentectomy and nucleoplasty by the same surgeon . the oswestry disability index ( odi ) questionnaire version 2.0 was used to assess degree of disability and functional status caused by sequestrated discs.4 ) all patients completed the odi questionnaire preoperatively and at 1-year postoperatively . in all cases , microscopic fragmentectomy was followed by intraoperative nucleoplasty . to quantify pain and functional outcomes , we used modified macnab 's criteria ( table 1 ) and a 10-point visual analog scale ( vas ) . statistical analysis was performed using the paired t - test and a confidence interval of 95% . a microscope ( carl zeiss , oberkochen , germany ) was mounted to aid removal of sequestrated disc material . exposure was obtained in each case according to the direction of the free fragment by making a 2 - 3 cm midline skin incision . in most cases , exposure of the ruptured disc fragment and involved nerve root was possible with minimal disturbance of the lamina or adjacent facet and limited bone removal . in all 24 patients , epidural fat was disturbed as less as possible and only bleeding epidural vessels were coagulated . intraoperative nucleoplasty was performed following successful microscopic fragmentectomy . under fluoroscopic guidance , a 17 guage , 6 inch cannula was inserted into the targeted disc space lateral to the facet joint . after confirming correct cannula placement , an introducer needle was inserted into the disc . once in proper position , of an introducer needle to the ruptured site a radiofrequency ( rf ) device ( l - disq , u&i company , uijeongbu , korea ) was inserted , and as suggested by a manufacture company guideline , at least 7 rf passes were performed . a microscope ( carl zeiss , oberkochen , germany ) was mounted to aid removal of sequestrated disc material . exposure was obtained in each case according to the direction of the free fragment by making a 2 - 3 cm midline skin incision . in most cases , exposure of the ruptured disc fragment and involved nerve root was possible with minimal disturbance of the lamina or adjacent facet and limited bone removal . in all 24 patients , epidural fat was disturbed as less as possible and only bleeding epidural vessels were coagulated . intraoperative nucleoplasty was performed following successful microscopic fragmentectomy . under fluoroscopic guidance , a 17 guage , 6 inch cannula was inserted into the targeted disc space lateral to the facet joint . after confirming correct cannula placement , an introducer needle was inserted into the disc . once in proper position , of an introducer needle to the ruptured site a radiofrequency ( rf ) device ( l - disq , u&i company , uijeongbu , korea ) was inserted , and as suggested by a manufacture company guideline , at least 7 rf passes were performed . the 24 patients ( mean age 41.2 years , range 20 to 74 years ) were followed for a minimum of 1 year ( mean follow - up 16.3 months ) . involved levels were ; l4 - 5 in 14 and l5-s1 in 10 . prior to the procedure , 11 patients had a disability score of 41 - 60% , 7 patients a score of 61 - 80% , 3 patients a score of 21 - 40% , and 3 patients a score of 81 - 100% ( table 2 ) . odis improved significantly after combined microscopic fragmentectomy and intraoperative nucleoplasty regardless of age group ( 20 - 40 , 41 - 60 , and > 60 ) , ( p<0.05)(table 3 ) . furthermore , according to the 10 point vas used ( 0=no pain and 10=intractable pain ) , mean pain levels dropped significantly from 8.31.5 to 2.61.6 ( p<0.05 ) . most patients ( 92% ) achieved an excellent or good result according to the modified macnab 's criteria at final follow - up ( excellent : 14 , good : 8) . however , one patient complained of occasional intermittent back and buttock pain , which subsequently required radiofrequency neurotomy , despite complete relief of radiating pain . two postoperative wound - related complications responded to antibiotics , and no recurrent disc herniation was encountered during follow - up . a free sequestrated fragment may migrate , which means that disc material moves away from the opening in the annulus.6,11 ) some migrated fragments will be sequestrated , but the term migrated refers to position and not to continuity.5 ) when the opening in a torn annulus is tiny and the intervertebral disc involved is relatively healthy , the optimal surgical approach is debatable . williams13 ) developed the concept of microsurgical removal as applied exclusively to offending material decompressing neural structures and the dural sac without touching relatively healthy discal or bony components or articulations ; this minimal approach has the advantage of leaving the normal anatomy relatively intact . young patients that present with radiologically preserved intervertebral height , a disc with otherwise little degenerative change , and a near intact posterior ligament and annulus fibrosus at operation are expected to profit most from fragmentectomy , and the small opening is expected to seal spontaneously and permanently without further herniation . wenger et al.11 ) also reported successful results for minimal bone removal and excision of only the completely extruded disc fragment without entering the disc space . however , this technique was not used in patients with a subligamentous herniation , a large tear in the annulus or in the pll , or with a fragment not clearly separated from the disc space . rogers8 ) reported a elevated recurrence rate when only the offending fragment was removed as compared with more extensive discectomies . unfortunately , complete clearance of all disc material is not possible , because there is no distinct border between the rigid annulus fibrosus and the soft nucleus pulposus , and thus , visualization of the intervertebral disc space and instrument accessibility are limited . furthermore , degenerative discopathy and extensive curettage of the interspace reduces intervertebral disc height and subsequently loosens ligaments and articular capsules . in addition , these loosenings , combined with reduced discal support in the anterior column , could result in segmental instability , facet arthrosis , and spondylosis , which in turn , could cause disc space narrowing and imbalanced weight bearing on spinal structures , and consequently , increase weight bearing at the facet joint.2,7,10 ) nucleoplasty , also known as percutaneous discectomy , is a minimally invasive procedure that uses rf energy to remove nuclear material.1 ) this technology involves the application of rf energy to a conductive medium and the creation of a highly focused plasma field ( composed of ionized particles ) around energized electrodes . thus , nucleoplasty combines coagulation and tissue ablation to form channels in nucleus and cause disc decompression . shabat et al.9 ) reported successful outcomes without complications in 87 patients with mechanical back and radicular pain at a minimal follow - up of 1 year after nucleoplasty . the best candidates for nucleoplasty are patients with radicular pain secondary to a contained disc herniation or axial pain . the findings of a cadaver study conducted by chen et al.1 ) showed that patients with incomplete annular tears and a minimally degenerated disc benefit most from nucleoplasty . accordingly , it appears patients with a large fissure are unlikely to benefit from nucleoplasty , despite " contained " disc herniation . instead , nucleoplasty could be alternative technique for small annular tear on behalf of the extensive microdiscectomy . although , sequestrated disc herniation tends to have large annular fissure with pll injury generally , we could obtain good surgical outcome by lowering intradiscal pressure without additional extensive discectomy . in the present study , significant improvements in vas scores and odis were achieved after a minimum follow - up of 1 year . furthermore , revision surgery due to re - herniation was not required during follow - up . however , there are always risks . infection , bleeding and neural injury are uncommon but possible risks . moreover , cuellar et al.3 ) reported progressive disc degeneration in patients with persistent pain after nucleoplasty in less than 1 year after surgery . it has been well established that patient selection is important for achieving satisfactory results after microscopic discectomy in cases of lumbar disc herniation . in their retrospective review of 259 lumbar discectomies . wera et al.12 ) showed a significant increase in revision surgery because of the re - herniation of extruded disc fragments through a large or massive annular defect . this suggests that microscopic fragmentectomy is performed for a sequestrated lumbar disc herniation without additional opening in case of small annular leak , it would help to ameliorate symptom . and however , despite the successful outcomes achieved , this by no means indicates that patients with sequestrated lumbar disc herniation should be treated using this technique . first , there is a lack of sufficiently powered studies and randomized control trials to support the widespread adoption of this procedure . second , we did not study the relation between the disc degeneration grade such as pfirrmann scores and clinical outcome . it should be performed cautiously due to its remarkable cost and the risk of neural injury , infection , bleeding and disc degeneration.3 ) in particular , despite our successful results , this does not mean most patients with lumbar disc herniation should be treated using this technique and patients with a large annular tear or pll injury should be included carefully . we suggest that a long - term randomized control comparative study should be conducted to confirm our results in the near future . combined microscopic fragmentectomy and intraoperative nucleoplasty without conventional discectomy is a safe operative modality for sequestrated lumbar disc herniation . as demonstrated by the present series , the results obtained are favorable , and thus , we recommend this procedure can be viewed as a alternative treatment as compared with standard microsurgical lumbar discectomy with extensive nucleus pulposus removal .

follistatin - like 3 ( fstl3 ) is a circulating glycoprotein , which is highly expressed in the reproductive system , but fstl3 is also expressed in adipose tissue , pancreas , liver , and skeletal muscle [ 2 , 3 ] . the only known function of fstl3 is to form high - affinity complexes with tgf- family members , most notably activin a and myostatin and thereby inhibiting their function [ 4 , 5 ] . previous human as well as animal studies indicate that the regulation of these two target proteins is altered with obesity , diabetes or both . thus elevated levels of activin a are found in adipose tissue from obese humans . moreover , patients with type 2 diabetes have elevated serum levels of activin a which correlate with markers of insulin resistance [ 7 , 8 ] . myostatin levels are elevated in serum from obese individuals and patients with type 2 diabetes have elevated myostatin expression in the skeletal muscle tissue . in prediabetic men , endurance training lowered myostatin in both serum and muscle . in line with the impact of obesity and diabetes on activin and myostatin , reports from both human and animal studies suggest that fstl3 is involved in glucose metabolism and obesity . thus , plasma fstl3 correlates inversely with the glucose concentration 1 hour after a 50 g oral glucose load in women whom later developed gestational diabetes . additionally , low plasma fstl3 levels are associated with insulin resistance during pregnancy in humans and low plasma concentrations of fstl3 in the first trimester are associated with an increased risk of developing gestational diabetes . in contrast , in obese leptin - deficient mice , fstl3 mrna is increased in the subcutaneous adipose tissue while fstl3 knockout mice have reduced visceral fat and hepatic steatosis . obesity and low - grade inflammation are tightly linked in humans [ 16 , 17 ] . inflammation may play a role in the regulation of fstl3 as the fstl3 promoter contains a nf-b binding site , and fstl3 protein is increased in response to tnf- stimulation in hepg2 cells . interestingly , activin a , which fstl3 inhibits , is a part of the proinflammatory response . the present study aimed at unravelling if circulating fstl3 is associated with and/or regulated by players in the metabolic syndrome , such as inflammatory mediators and insulin . therefore , we studied fstl3 in a human cross - sectional study including healthy controls , individuals with impaired glucose tolerance , and patients with type 2 diabetes . given that tnf- stimulates fstl3 in vitro and given the elevated levels of tnf- in patients with obesity and insulin resistance , we measured fstl3 in response to e. coli lipopolysaccharide ( lps ) , tnf- , and il-6 infusions in humans . we hypothesized that fstl3 in humans would increase with insulin resistance and/or obesity and in response to inflammation . here we demonstrate that fstl3 is elevated in obese subjects and that fstl3 increases in response to inflammation . data from the cross - sectional part of this paper has previously been published with data from 187 subjects . in the present study plasma fstl3 levels the reason for the difference between the present and the previous papers with regard to n - value is that 13 persons were excluded in the first publication based on cognitive function . accordingly , we included all 200 subjects . in brief , subjects who participated in the study were recruited to the cross - sectional study in order to obtain three groups representing different levels of glucose tolerance : a group with normal glucose tolerance ( con ) , a group with impaired glucose tolerance ( igt ) , and a group of subjects with type 2 diabetes mellitus ( t2 dm ) . the three groups were defined based on the oral glucose tolerance test ( ogtt ) according to who criteria . insulin sensitivity and pancreatic beta - cell function were calculated by using the homeostatic model assessment ( homa s and homa b ) as described . participants were allowed to take oral antidiabetic drugs but had to stop 1 week prior to the ogtt . after an overnight fast the participants reported to the laboratory for a general health examination ; a blood sample was taken for measurement of fasting glucose , insulin c - peptide , tnf- , il-6 , crp , leptin , and adiponectin . dual - energy x - ray absorptiometry ( dxa ) was used to determine fat and lean mass as described in . the participants were recruited by advertisement in local newspapers , and 3 subjects were recruited from the steno diabetes centre database . exclusion criteria were treatment with insulin , recent or ongoing infections , history of malignant cancer and severe chronic inflammatory diseases , fasting glucose above 12 mmol / l , and a blood pressure above 180 mm hg/110 mm hg . samples were retrieved from a study on the inflammatory response to lps in t2 dm compared to normal glucose tolerance [ 2124 ] . originally , the material included 23 healthy control subjects and 19 patients with type 2 diabetes . only samples from 12 healthy controls and 11 subjects with type 2 data on cytokine levels in response to lps is shown in ( figures 1(a)1(c ) ) . in brief , subjects reported in the laboratory at 08:00 in the morning after an overnight fast and rested in the supine position while having catheters placed in antecubital veins for injection and blood sampling . an intravenous injection of escherichia coli lps ( lot g2 b274 , united states pharmacopeial convention , rockville , md , usa ) was administered at a dose of 0.3 ng / kg . plasma fstl3 was measured at time points 0 , 2 4 , 6 , and 8 hours . only samples from 8 volunteers were available for the present study . in brief , subjects reported to the laboratory at 08:00 in the morning after an overnight fast and rested in the supine position while having catheters placed in antecubital veins for infusion and blood sampling . subjects received a 4-hour infusion of rhtnf-(beromun ) 700 ng / mh ( boehringer - ingelheim , biberach an der riss , germany ) and on a separate day a 4-hour infusion of 20% albumin ( tnf- vehicle ) . the two study days were separated by at least one week . during the tnf- infusion plasma tnf- level was elevated to approximately 16 pg / ml and remained constant during the 4 hours of infusion as shown in ( figure 1(a ) ) . fstl3 was measured at time points 2 , 0 , 1 , 2 , 3 , and 4 hours . six healthy male subjects participated in the study . in brief , subjects underwent a general health examination before participating . subjects reported in the laboratory at 08:00 in the morning after an overnight fast and rested in the supine position while having catheters placed in antecubital veins for infusion and blood sampling . subjects received a 3-hour infusion of rhil-6 ( sandoz , basle , switzerland ) at a rate of 5 ug / h . during the infusion of rhil-6 plasma il-6 level was elevated to approximately 150 pg / ml as shown in ( figure 1(a ) ) . fstl3 was measured at time points 0 h , 1 h , 2 h , 3 h , 6 h , and 24 h. six healthy men aged 28.8 2.5 years ( mean sd ) and bmi 22 0.6 kg / m ( mean sd ) were recruited through local newspapers . the participants reported in the laboratory at 08:00 the morning after an overnight fast and rested in the supine position . two peripheral intravenous catheters were placed : one for infusion of insulin and glucose and one for blood sampling . subjects received continuous 5-hour infusion of insulin ( actrapid , 100 iu / ml ; novo nordisk , bagsvaerd , denmark ) at 0.05 u min m. blood glucose was measured at bedside and glucose was infused by a computer - controlled infusion pump at rates adjusted according to blood glucose levels in order to uphold euglycemia . fstl3 was measured in plasma from samples at 15 minutes before the start of the clamp and at 45 min , 1 h 45 min , 2 h 45 min , 3 h 45 min , and 4 h 45 min relative to the start of the clamp . all studies were approved by the ethical committee and performed in accordance with the helsinki declaration . written consent was obtained from each subject prior to enrolment in the study . in all studies , plasma obtained after lps and tnf- study , fstl3 was measured using an fstl3 duo kit from cat number dy 1288 from r&d systems . this kit has previously been used by others and at the time of analysis the only available kit . the kit was used according to the manufacturer 's instructions . in the cross - sectional study , the il-6 infusion study , and the insulin clamp study , fstl3 was measured using a newer kit from human flrg ( quantikine elisa kit dflrg0 ) from r&d systems . calculation of interassay variation was done by having two different control samples ( in duplicate ) on each plate . mean value of fstl3 was calculated for each control , for the 6 plates used to measure fstl3 in the cross - sectional study . intra - assay was calculated as the average cv for all the duplicates on each plate , by all 6 plates given a cv of 2.8% for the newest kit . interassay variation cv% for the kit used in the lps and the tnf- studies was 3.8% calculated for three plates . intra - assay variation calculated for the three plates used in the lps study was 4.0% . plasma adiponectin , both high and low molecular forms , and leptin levels were measured using an elisa kit from msd ( meso scale discovery , gaithersburg , usa ) according to the manufacturer 's instructions . interassay variability was , 9% for leptin , and 17% for adiponectin , and intra - assay variability was 4.7% for leptin and 3.4% for adiponectin . as in the original cross - sectional study data is presented as median and 25th + 75th quartiles . as many of the variables were not normally distributed , nonparametric tests were used for statistical analysis . to test for differences between the 3 glycemic groups , controls , igt , and type 2 diabetics , kruskall - wallis test was used , and when significantly different , wilcoxon rank test was applied to test con versus igt and con versus t2 dm corrected for multiple testing using bonferroni correction . for differences between obese and nonobese , or between females and males , a wilcoxon rank - sum test ( two - sample ) was applied . all analyses on the cross - sectional study were done using sas software version 9.1 ( sas institute ) . in all infusion studies , plasma fstl3 is presented as geometric mean standard error of the mean ( sem ) . the effect of time and group on fstl3 by lps and tnf- infusion was assed using two - way anova . the effect of il-6 and hyperinsulinemia was assessed by one - way anova using the proc mixed procedure , sas 9.1 . as a post hoc test as compared to healthy controls , patients with t2 dm were older and had higher fasting insulin , glucose , c - peptide , and il-6 levels , and lower adiponectin , homa s , homa b , and vo2max. subjects with igt also had elevated fasting insulin , glucose , c - peptide , il-6 , and tnf- levels and lower homa s and adiponectin levels compared to con . in the total study population of 200 subjects , fstl3 did not differ between con ( 6216 ( 54476884 ) pg / ml ) , igt ( 6344 ( 57337319 ) pg / ml ) , and t2 dm groups ( 6062 ( 51117503 ) pg / ml ) . kg / m ) compared to nonobese subjects , both in the total group 6892 pg / ml ( 60317888 pg / ml ) versus 5751 pg / ml ( 50816388 pg / ml ) and within subgroups . in all subjects combined ( n = 200 ) , fstl3 correlated positively with age , weight ( p < 0.001 ) , hip ( p < 0.001 ) and waist ( p < 0.001 ) circumference , bmi ( p < 0.001 ) , whole body fat ( p < 0.001 ) , and trunk fat mass ( p < 0.001 ) and negatively with height ( p < 0.05 ) and vo2max ( p < 0.001 ) ( table 2 ) . fstl3 also correlated with weight , hip and waist circumference , bmi , whole body fat , and trunk fat mass in individual groups . in the nonobese subjects , fstl3 was positively correlated with age , whole body fat , fat - free mass , and trunk fat . plasma fstl3 correlated with age , hip and waist circumferences , bmi , whole body fat , trunk fat , fat - free mass lean body mass , and fat - free mass and negatively with vo2max in obese subjects . further analyses showed that plasma fstl3 was correlated with fasting plasma insulin , fasting c - peptide , and -cell function assessed by homa b , but not with fasting glucose . fstl3 correlated negatively with insulin sensitivity measured by homa s in all groups and in nonobese , but not in obese subjects . in all subjects combined , plasma fstl3 correlated with plasma tnf- and il-6 ( p < 0.001 ) . in patients with t2 dm , fstl3 correlated with both tnf- and il-6 , whereas fstl3 only correlated with il-6 and not tnf- in the control and the igt groups ( table 2 ) . fstl3 correlated with leptin in all subjects and subgroups . no correlation was observed to adiponectin . females have higher circulating fstl3 compared to males plasma fstl3 was higher in females 6618 ( q25 5769q75 7429 ) pg / ml n = 100 than in males , 5892 ( q25 5205q75 6824 ) pg / ml n = 100 ; ( p = 0.0024 ) . this differences were only evident in obese ( p = 0.002 ) , but not between nonobese females and males ( p = 0.083 ) ( data not shown ) . as stated in table 1 gender distribution did not differ between the different glycemic groups or was there a different in gender distribution between obese and nonobese subjects ( p = 0.6712 chi - square ) . as plasma fstl3 correlated with markers of low - grade inflammation , we investigated if plasma fstl3 was acutely regulated by inflammation . in the lps study [ 21 , 23 ] , patients with type 2 diabetes and healthy controls were administered an lps bolus of 0.3 ng / kg , which induced an inflammatory response with increased body temperature , heart rate , neutrophil count , and tnf- and il-6 levels . in both groups , lps increased circulating fstl3 over the 8-hour intervention period , with the highest levels observed after 6 hours . the response to lps did not differ between controls and patients with t2 dm ( figure 2(a ) ) . next , we investigated if plasma fstl3 was regulated directly by the proinflammatory cytokines tnf- or il-6 . during tnf- infusion plasma fstl3 increased significantly after 3 hours and increased further at 4 hours ( figure 2(b ) ) . no acute effect on plasma fstl3 was observed during il-6 infusion ( figure 2(c ) ) . however , the 24-hour time point was modestly but significantly elevated compared to all other time points . as fstl3 was strongly correlated to fasting insulin , we investigated the direct fstl3 response to a hyperinsulinemic euglycemic clamp . in the present study , the plasma level of fstl3 is elevated in obese subjects independent of glycemic status and is associated with fat mass as well as with plasma tnf- , a strong marker of inflammation . moreover , fstl3 is directly induced by both lps and tnf- administration , indicating that systemic inflammation increases fstl3 and that this effect is mediated by tnf-. finally , although plasma fstl3 is associated with markers of glucose intolerance , fstl3 is not increased in igt or t2 dm , suggesting an indirect rather than a direct relationship between insulin resistance and fstl3 . a hyperinsulinemic clamp failed to increase fstl3 in plasma , suggesting that this putative relationship is not mediated by insulin . activin a is modestly increased in plasma from type 2 diabetic patients and correlated with clinical parameter of type 2 diabetes . in continuation , a 35% increase in serum myostatin is observed in obese humans . furthermore , training results in a 22% reduction in serum myostatin in middle age men . thus , the 20% increase in plasma fstl3 reported here in obese compared to nonobese subjects indicates that regulation of the myostatin / activin a fstl3 system in humans results in relatively small changes in circulating levels . . thus elevated levels of fstl3 have been observed in women with preeclampsia [ 29 , 30 ] and low levels of plasma fstl3 have been reported in women with gestational diabetes [ 12 , 14 , 29 ] . the expression of fstl3 in placenta is increased and decreased , respectively [ 14 , 29 , 30 ] . this suggests that the placenta may contribute to circulating fstl3 during pregnancy making comparison to type 2 diabetes and obesity difficult . in line with our data , others have demonstrated that fstl3 correlates with age and body weight and bmi . the observation that fstl3 correlated with age may be related to the increase in the ligands of fstl3 , activin a and myostatin , which are increased with age [ 19 , 33 ] . in the present study plasma fstl3 is not different between controls , igt , or t2 dm and shows no association with fasting glucose suggesting that fstl3 is not involved in insulin resistance . furthermore plasma fstl3 is not increased in response to insulin , indicating that fstl3 is not regulated by insulin . we did find a positive correlation between plasma fstl3 and fasting insulin , c - peptide and a negative correlation to insulin sensitivity ( homa s ) . low plasma fstl3 is associated with greater risk of developing gestational diabetes as fstl3 correlates negatively with peak glucose levels during a 50 g oral glucose load . in another report also on gestational diabetes , no correlation to insulin resistance ( homa ir ) the difference between our data and the gestational diabetes studies may be explained by contribution of fstl3 to the circulation by the placenta during pregnancy . furthermore , this could indicate that low fstl3 and thereby a potentially higher myostatin and activin a levels in plasma could be involved in the development of insulin resistance . in support of this view is the observation that activin a but not myostatin is increased in gestational diabetes . in the present study plasma fstl3 is elevated in obese subjects and fstl3 is positively associated with total fat mass , trunk fat mass , and circulating leptin . moreover , fstl3 is expressed in adipocytes and is increased in obese mice and knockout of fstl3 in mice alters fat distribution . this is further supported by the finding that females , who generally have both higher fat percentage and leptin levels , also have higher fstl3 in plasma . as mentioned women with preeclampsia leptin is also expressed by the placenta and plasma leptin levels are also increased in preeclampsia , which could indicate a common source or similar regulation of leptin and fstl3 . age is a possible confounder in the present study , as age is positively associated with plasma fstl3 in both nonobese and obese ( table 3 ) . however nonobese subjects were older compared to the obese group ( data not shown ) . this pattern was also present when the three glycemic groups were divided by obesity ( data not shown ) . in the nonobese and obese groups 25 and 20 subjects were in treatment with statins , respectively . therefore it is unlikely that plasma fstl3 is increased in the obesity group due to age or lipid lowering drugs . exploring the association between fstl3 and low - grade inflammation observed in the cross - sectional study , by acute administration of lps and tnf- , we observed an increase of plasma fstl3 levels in humans . activin a is well known for its acute proinflammatory actions as part of the innate immune system in response to lps administration . as fstl3 antagonizes activin a , the increase in fstl3 may potentially be considered a countercompensatory response to control the inflammatory process . this interpretation is consistent with the time course for plasma fstl3 in both the lps and the tnf- studies where fstl3 peaks later than activin a . il-6 does not acutely regulate fstl3 consistent with that il-6 is not the driver for fstl3 during the lps bolus . fstl3 is increased after the il-6 infusion at 24-hour time point compared with all other time points . however , it is possible that il-6 may increase fstl3 levels during chronic conditions such as obesity or low - grade inflammation . due to the mainly cross - sectional design of the study , we can not distinguish if obesity per se or low - grade inflammation is the main driver for the increased fstl3 in obese subjects . neither are we able to clarify which tissue or tissues are responsible the circulating levels of fstl3 during obesity or inflammation . we have demonstrated that circulating levels of fstl3 are increased in obese subjects and that high fstl3 levels are associated with fat mass and markers of low - grade inflammation . the finding that plasma fstl3 was increased in response to tnf- , but not il-6 , infusion suggests that tnf- is an inflammatory driver of increased systemic levels of fstl3 .

cancer of the external auditory canal ( eac ) and middle ear is rare , with a prevalence of one to six patients per 1 million people [ 1 - 3 ] . although several treatment modalities have been investigated , there are no standard guidelines for treating cancers of the eac and middle ear due to a lack of prospective randomized studies . a surgical resection with a clear tumor margin is usually recommended for resectable disease . on the other hand , the role of adjuvant and definitive radiotherapy ( rt ) in treating unresectable disease is less well - defined . the prognosis of an eac carcinoma depends on the disease stage and the primary treatment approach used . investigators have reported the 5-year survival rates ranging from 10% for advanced stage to as high as 85% for early stage disease [ 4,6 - 9 ] . in the literature , the 5-year locoregional control rates range from 20% for advanced stage to 70% for early stage disease . as there is no widely accepted staging system , using the results from different reports to compare the treatment outcomes can be difficult . moreover , although several articles describe the efficacy of rt for eac tumors , there is little information on the relationships between the extent of disease and the clinical outcomes for different types of rt . to help fill this void , this study evaluated the treatment outcomes for 32 patients with a carcinoma of the eac and middle ear who were treated with postoperative or definitive rt , with or without chemotherapy , at the authors institution over a 20-year period . the records of 32 patients treated with postoperative or definitive rt at the yonsei cancer center for primary carcinoma of the eac and middle ear between 1990 and 2013 were reviewed retrospectively . . 4 - 2015 - 0884 ) approved this retrospective study in accordance with the ethical guidelines and the declaration of helsinki . all patients had received computed tomography ( ct ) or magnetic resonance imaging ( mri ) scans , or both , prior to treatment . four , eight , five , and 15 patients were classified as having stage i ( t1n0 ) , stage ii ( t2n0 ) , stage iii ( t3n0 ) , and stage iv ( t3n1 , t4n0 , t4n1 ) carcinoma , respectively . early stage carcinoma and advanced stage carcinoma was defined as t1/t2 ( n=12 ) and t3/t4 or n positive ( n=20 ) , respectively . surgical procedures were chosen at the discretion of the surgeon in accordance with the tumor extent and nodal status . six of these patients received a total or subtotal temporal bone resection ( tbr ) , eight received a partial or lateral tbr , and seven received a wide excision . postoperative rt was used to treat 21 ( 65.6% ) patients and definitive rt was used to treat 11 ( 34.4% ) . all patients were treated using external beam rt with curative intent . with the exception of two patients treated before the year 2000 the total doses applied to the primary tumor bed were 59.4 or 60 gy for patients with negative tumor margins and between 54 and 71.4 gy for those patients with positive tumor margins . the prescribed doses in definitive rt were 51 to 75.6 gy at 1.8 to 2.5 gy per fraction for the primary tumor bed . based on the pretreatment ct or mri scan results , the treatment volumes were drawn to include the primary tumor bed or ipsilateral lymph nodes , or both , with 1 - 2 cm margins . the differences in patient characteristics between the two radiation groups were assessed using a chi - square test . the overall survival ( os ) rates , disease - free survival ( dfs ) rates , disease control rates , and failure patterns were evaluated . the survival time was defined as the interval from the date of the completion of rt to the date of the last follow - up or death . the records of 32 patients treated with postoperative or definitive rt at the yonsei cancer center for primary carcinoma of the eac and middle ear between 1990 and 2013 were reviewed retrospectively . . 4 - 2015 - 0884 ) approved this retrospective study in accordance with the ethical guidelines and the declaration of helsinki . all patients had received computed tomography ( ct ) or magnetic resonance imaging ( mri ) scans , or both , prior to treatment . four , eight , five , and 15 patients were classified as having stage i ( t1n0 ) , stage ii ( t2n0 ) , stage iii ( t3n0 ) , and stage iv ( t3n1 , t4n0 , t4n1 ) carcinoma , respectively . early stage carcinoma and advanced stage carcinoma was defined as t1/t2 ( n=12 ) and t3/t4 or n positive ( n=20 ) , respectively . surgical procedures were chosen at the discretion of the surgeon in accordance with the tumor extent and nodal status . six of these patients received a total or subtotal temporal bone resection ( tbr ) , eight received a partial or lateral tbr , and seven received a wide excision . postoperative rt was used to treat 21 ( 65.6% ) patients and definitive rt was used to treat 11 ( 34.4% ) . all patients were treated using external beam rt with curative intent . with the exception of two patients treated before the year 2000 the total doses applied to the primary tumor bed were 59.4 or 60 gy for patients with negative tumor margins and between 54 and 71.4 gy for those patients with positive tumor margins . the prescribed doses in definitive rt were 51 to 75.6 gy at 1.8 to 2.5 gy per fraction for the primary tumor bed . based on the pretreatment ct or mri scan results , the treatment volumes were drawn to include the primary tumor bed or ipsilateral lymph nodes , or both , with 1 - 2 cm margins . the differences in patient characteristics between the two radiation groups were assessed using a chi - square test . the overall survival ( os ) rates , disease - free survival ( dfs ) rates , disease control rates , and failure patterns were evaluated . the survival time was defined as the interval from the date of the completion of rt to the date of the last follow - up or death . the median age was 51 years ( range , 26 to 87 years ) at the time of diagnosis . the primary tumor site was the eac and middle ear in 31 and one patient , respectively . a histological examination revealed squamous cell carcinoma ( scc ) in 21 patients ( 65.6% ) . the other histological types present in the other patients were adenoid cystic carcinoma ( acc , 28.1% ) , adenocarcinoma ( 3.1% ) , and malignant melanoma ( 3.1% ) . most of the patients had eastern cooperative oncology group performance scores of 0 or 1 . the most frequent symptoms and signs were otorrhea ( n=12 , including two patients with hemorrhagic otorrhea ) , preauricular mass ( n=8 ) , otalgia ( n=7 ) , and facial palsy ( n=5 ) . an analysis of the treatment patterns showed that 59% ( n=19 ) of the patients had been treated with combined surgery and rt , 22% ( n=7 ) with chemoradiotherapy , and 19% ( n=6 ) with rt alone . nine of the 12 patients with stage i - ii disease received surgery with rt ; three patients received rt alone . ten of the 20 patients with stage iii - iv disease underwent surgery with rt ; seven underwent combined chemoradiotherapy and three were treated using rt alone ( fig . the median post - rt follow - up period was 51 months ( range , 7 to 286 months ) . twenty - four of the 32 patients ( 75% ) experienced a complete response after rt ; five patients ( 15.6% ) had a partial response . the 5-year os and the 5-year os and dfs rates for early stage versus advanced stage disease were 70.7% versus 48% ( p=0.315 ) and 65.6% versus 41.4% ( p=0.435 ) , respectively . the 5-year os rate was 64.8% and 42.4% for the postoperative and definitive rt group , respectively ( p=0.261 ) . the 5-year dfs rate was 49.1% for the postoperative rt group and 62.2% for the definitive rt group ( p=0.835 ) . sixteen patients ( 50% ) showed no evidence of a disease status without recurrence at the last follow - up visit . the disease control rates for patients with early stage versus advanced stage carcinoma were 55.6% ( 5/9 ) versus 50% ( 6/12 ) in the postoperative rt group and 66.7% ( 2/3 ) versus 37.5% ( 3/8 ) in the definitive rt group , respectively . eleven patients ( 52.4% ) and five patients ( 45.5% ) in the postoperative rt and definitive rt group , respectively , were considered cured . two patients experienced disease progression after the initial treatment , and were excluded from the analysis of the failure patterns . overall , 14 patients ( 46.7% ) experienced treatment failure ; four of these were classified as local failure and one as regional failure . the most common metastatic site was the lung ( seven cases ) , followed by the spine ( two cases ) and bone ( one case ) . table 3 lists the results of an analysis of the failure patterns according to the rt type . rate was 52.4% ( 11/21 ) in the postoperative rt group and 44.4% ( 4/9 ) in the definitive rt group . the median age was 51 years ( range , 26 to 87 years ) at the time of diagnosis . the primary tumor site was the eac and middle ear in 31 and one patient , respectively . a histological examination revealed squamous cell carcinoma ( scc ) in 21 patients ( 65.6% ) . the other histological types present in the other patients were adenoid cystic carcinoma ( acc , 28.1% ) , adenocarcinoma ( 3.1% ) , and malignant melanoma ( 3.1% ) . most of the patients had eastern cooperative oncology group performance scores of 0 or 1 . the most frequent symptoms and signs were otorrhea ( n=12 , including two patients with hemorrhagic otorrhea ) , preauricular mass ( n=8 ) , otalgia ( n=7 ) , and facial palsy ( n=5 ) . an analysis of the treatment patterns showed that 59% ( n=19 ) of the patients had been treated with combined surgery and rt , 22% ( n=7 ) with chemoradiotherapy , and 19% ( n=6 ) with rt alone . nine of the 12 patients with stage i - ii disease received surgery with rt ; three patients received rt alone . ten of the 20 patients with stage iii - iv disease underwent surgery with rt ; seven underwent combined chemoradiotherapy and three were treated using rt alone ( fig . the median post - rt follow - up period was 51 months ( range , 7 to 286 months ) . twenty - four of the 32 patients ( 75% ) experienced a complete response after rt ; five patients ( 15.6% ) had a partial response . the 5-year os and dfs rates for all patients were 57% and 52% , respectively . the 5-year os and dfs rates for early stage versus advanced stage disease were 70.7% versus 48% ( p=0.315 ) and 65.6% versus 41.4% ( p=0.435 ) , respectively . the 5-year os rate was 64.8% and 42.4% for the postoperative and definitive rt group , respectively ( p=0.261 ) . the 5-year dfs rate was 49.1% for the postoperative rt group and 62.2% for the definitive rt group ( p=0.835 ) . sixteen patients ( 50% ) showed no evidence of a disease status without recurrence at the last follow - up visit . the disease control rates for patients with early stage versus advanced stage carcinoma were 55.6% ( 5/9 ) versus 50% ( 6/12 ) in the postoperative rt group and 66.7% ( 2/3 ) versus 37.5% ( 3/8 ) in the definitive rt group , respectively . eleven patients ( 52.4% ) and five patients ( 45.5% ) in the postoperative rt and definitive rt group , respectively , were considered cured . two patients experienced disease progression after the initial treatment , and were excluded from the analysis of the failure patterns . overall , 14 patients ( 46.7% ) experienced treatment failure ; four of these were classified as local failure and one as regional failure . the most common metastatic site was the lung ( seven cases ) , followed by the spine ( two cases ) and bone ( one case ) . table 3 lists the results of an analysis of the failure patterns according to the rt type . rate was 52.4% ( 11/21 ) in the postoperative rt group and 44.4% ( 4/9 ) in the definitive rt group . primary cancer of the eac and middle ear is extremely rare and tends to be locally advanced at presentation and has a poor prognosis . surgery ( e.g. , lateral tbr for t1/t2 disease and subtotal or total tbr for t3/t4 disease ) is generally the recommended treatment . in most clinical settings , however , less invasive surgeries are undertaken to treat this disease , even when t3/t4 disease is present , because the eac is located adjacent to the critical organs , such as the brain and major vessels . nevertheless , the use of less invasive surgical techniques is the main reason for local failure after surgical treatment . a comparison of the treatment strategies for eac and middle ear cancer is hindered by treatment and staging classification heterogeneity due to the small patient populations in published series . according to the pittsburgh staging system , standard therapies include lateral tbr or definitive rt for t1 eac tumors , lateral tbr combined with postoperative rt for t2 tumors , and subtotal or total tbr with postoperative rt for t3 and t4 tumors . on the other hand , despite these standardized treatment recommendations , the prognoses remain poor . at a single institutional experience , seven patients were treated using definitive rt and 22 patients were treated with postoperative rt . better local and local - regional control was achieved for the early stage ( t1/2 ) tumors than for the late - stage ( t3 ) tumors , even though less than half of the patients were cured . this suggests that a suboptimal treatment approach is being used for the local management of eac cancers . the use of meta - analysis is important for an optimal evaluation of the treatment strategies because it has been difficult for a single institution to obtain a sufficient number of cases to achieve satisfactory statistical power . a review of 26 publications containing information on 144 patients showed that patients with carcinoma confined to the eac had similar survival times , regardless of the type of surgery performed ( e.g. , mastoidectomy , lateral tbr , and subtotal tbr ) . patients with an extension of the disease into the middle ear had longer survival times if they received a subtotal tbr . on the other hand , the addition of radiation therapy to lateral tbr did not improve the survival rates . another multi - institutional review of 87 records that focused primarily on the roles of surgery and rt in these patients found that the 5-year os and dfs rates for all patients were 55% and 54% , respectively . the 5-year dfs rates for the t1 , t2 , and t3 patients were 83% , 45% , and 0% in the rt group , and 75% , 75% , and 46% in the surgery with rt group , respectively . they concluded that a radical rt is the treatment of choice for stage t1 , and surgery combined with rt is recommended for t2 or t3 stage cancer . many authors have suggested that postoperative rt is effective for the control of residual tumors at incomplete resection margins . the present study compared treatment outcomes between early stage and advanced stage tumors according to the treatment modality ( e.g. , postoperative and definitive rt ) . although the results were not statistically significant , they suggested that the prognosis of patients with early stage tumors is more favorable than for patients with advanced stage tumors . these results also indicated that patients with early stage cancer achieve better outcomes with definitive rt rather than postoperative rt . the stage - related disease control rates for definitive and postoperative rt were 66.7% and 55.6% , respectively . therefore , this study suggests that patients with early stage cancer can undergo less invasive treatment . some authors have suggested that small lesions without bony erosion can be treated with rt alone . on the other hand , the disease control rates for advanced stage tumors treated with postoperative and definitive rt were 50% and 37.5% , respectively . therefore , patients with advanced stage tumors show better outcomes when postoperative rt is used . although the optimal radiation dose is unclear , the median doses for tumor control in this study were 59.4 gy for postoperative rt and 68.4 gy for definitive rt . a previous study indicated that total doses of 65 to 75 gy are needed to control the disease . one limitation of this study was that it was a retrospective , single institution study that included a 20-year time period . heterogeneity among the patient characteristics ( e.g. , tumor histologic type ) might have confounded the results . the most common histologic type of carcinoma of the eac and middle ear is scc . this type was also the most common type ( 65.6% ) in the present study population . acc is a special entity among tumors of the eac ; it has a slow proliferation rate and a low tendency towards lymphatic spread . on the other hand , lesions recur frequently when they are not excised completely , which contributes to the poor prognosis of acc patients . therefore , more aggressive surgery is recommended for patients with incompletely resected acc and patients with late - stage diseases . paradoxically , this study revealed 5-year os rates of 76.2% and 50% for patients with acc and scc , respectively . secondly , although the mri findings can be used to evaluate the parotid , soft tissue , infratemporal fossa , and temporal dura mater involvement , the patients in the present study were staged according to the clinical and ct - based radiologic findings so that the results would be comparable to those in other studies that used the pittsburgh staging system . thirdly , 14 patients ( 46.7% ) experienced treatment failure ; the incidence of distant failure was particularly high . physicians from several institutions have applied chemoradiation therapy ( crt ) to improve the patients prognosis . the results of meta - analysis to assess the role of crt for eac scc suggested that preoperative crt may improve the survival of surgically treated patients with eac scc . the meta - analysis results also suggested that definitive crt may be an effective alternative to surgical resection . surgical approaches to treat advanced stage eac and middle ear cancer often cause severe complications . in addition , because the recurrence rates are high , a minimum of 66 gy is recommended for patients with positive margins . newer treatment techniques , such as 3-dimensional conformal and intensity - modulated rt , can be used to deliver higher doses to the tumor and lower doses to the surrounding critical organs . patients with early stage eac and middle ear carcinoma had better outcomes when definitive rt was used . the high rate of distant failure after rt , with or without surgery , reflected the lack of a consensus on the best therapeutic approach for treating carcinomas of the eac and middle ear .

the effects of central nervous system ( cns ) injury or disease include altered sensation and pain and impaired control of movement , paralysis and spasticity . contemporary emergency and rehabilitative medical care has developed to successfully prevent and treat secondary medical complications of neurological disorders providing a near - normal life expectancy for people with neurological damage . medicine at present , however , can not anatomically repair the affected parts of the cns to return normal neurological function . this results in a large population of patients who must endure chronic sensory and motor disabilities and need solutions that improve their quality of life . to enhance recovery from cns injury or disease , restorative neurology is a discipline that works through the modification of residual , altered , yet remaining nervous system function . the therapeutic paradigm was the modulation of cns activity rather than the irreversible ( destructive ) modification of the cns structure . an example of this development was vladimir liberson 's functional electrotherapy , later renamed functional electrical stimulation ( fes ) , for patients with foot drop after stroke . the availability of portable stimulators further led to multi - site fes to generate standing and some stepping - movements in paraplegic patients and to augment gait rehabilitation in patients with incomplete spinal cord injury ( sci ) . in 1967 , norman shealy implanted the first neuroaugmentive device for the relief of intractable pain which he termed dorsal column stimulator . the potential of this technique of spinal cord stimulation ( scs ) in movement disorders was recognized soon thereafter . the commercial availability of implantable stimulators paved the way for the current neuromodulation therapies for altered sensation and pain , peripheral and cardiac ischemia , severe depression and psychiatric conditions . in movement disorders , contemporary neuromodulation therapies target brain structures , cranial nerves , spinal cord , and peripheral nerves and influence the nervous system activity by the interaction of an electrical or neurochemical input with specific neural circuits [ 57 ] . deep brain stimulation with implanted leads is applied in parkinson 's disease , in dystonia and as a treatment for essential tremor . vagus nerve stimulation via an implanted electrical stimulator is applied in the treatment of intractable epilepsy . electrical epidural scs can alleviate various disorders of the motor system in patients with sci , multiple sclerosis ( ms ) , cerebral palsy , and people with post - traumatic and post - stroke brain injury . sacral nerve stimulation with an implanted lead , also termed sacral neuromodulation , is an accepted treatment method for urinary pelvic disorders and incontinence . the development of implantable drug pumps allowed for the chronic delivery of medication to the physiological target site of the spinal cord to modulate motor function . intrathecal delivery of baclofen is well - established in the treatment of spinal spasticity caused by ms or sci and spasticity of cerebral origin . methods for the restoration of the neuromuscular lower limb functions will be the focus of the present review . these approaches utilize the intact peripheral nerves and the capacity of the spinal cord of neural signal processing that remain after impairment of the cns . then , scs for the improvement of lower limb function in motor disorders ( primarily ) with spinal cord involvement will be discussed , i.e. the improvement of spinal spasticity and altered motor control due to ms and sci . these topics will lead to the report on our human work of epidural and transcutaneous stimulation specifically targeting the lumbar spinal cord for enhancing motor functions in people with sci , supplemented by pertinent human research of other investigators . the reviewed literature was partially selected following the development process of the recently published monograph on restorative neurology of motor control after sci , and from a comprehensive reference work encompassing the various aspects of neuromodulation . the first modern applications of fes to generate movements of paralyzed lower limbs in sci and hemiplegic subjects were reported in the early 1960s . fes is applied to produce patterned muscle contractions and movements for specific function in people with motor control impaired by upper motor neuron dysfunction . fes methodologies use timed sequences of short bursts of electrical pulses , rather than sustained stimulation commonly applied in other neuromodulation therapies . muscles are either activated by electrical stimulation of the respective lower motoneurons , usually near the corresponding motor endplate region , or by peripheral afferent stimulation and the resulting spinal reflex . fes systems for the restoration of lower limb movement can be divided into applications for the facilitation of ankle dorsiflexion in hemiplegia and the generation of standing and stepping - movements in sci individuals . both , surface stimulators and implantable devices have been developed . in the therapeutic treatment of foot drop after stroke , footswitch- or tilt sensor - triggered peroneal nerve stimulation elicits a flexion reflex that generates hip , knee , and ankle movements and enhances the swing phase during gait . a review that evaluated seven case series and one randomized clinical trial of fes in stroke patients with foot drop suggested a positive effect of fes on walking speed , with a pooled improvement by 38% . commercially available and fda approved surface stimulation systems primarily designed for foot drop correction are the walkaide system ( innovative neurotronics inc . , austin , tx ) , the odstock dropped foot stimulator ( odstock medical limited , salisbury district hospital , salisbury , uk ) , and the ness l300 foot drop system ( bioness inc . , valencia , ca ) . implantable fes devices were designed for more selective control of the evoked muscle activities by stimulating separate groupings of fibers within a nerve trunk . the actigate system ( neurodan a / s , aalborg , denmark ) is based on an implanted cuff electrode with four channels positioned to activate different nerve fibers within the common peroneal nerve and can elicit dorsal and plantar flexion as well as inversion and eversion . safety and the effect on walking of the actigate system in hemiplegia were evaluated in a phase ii trial . the authors reported long - term improvements in walking speed by 19% and in the distance walked in 4 min . the stimustep ( finetech medical ltd , welwyn garden city , uk ) is a two - channel implantable peroneal nerve stimulator . randomized controlled trials of the effects of the stimustep in hemiplegia demonstrated an increased walking speed by 23% and improvements of spatiotemporal gait parameters and kinematics . apart from the immediate effect of stimulation , training with the fes system can ( temporarily ) enable patients to dorsiflex their foot volitionally to ambulate with less fatigue . in paraplegic individuals with thoracic sci , multi - site fes can generate standing and short - distance ambulation by stimulating the quadriceps , sometimes together with the gluteal muscles , to produce lower limb extension during stance phases , and the peroneal nerve to initiate a swing - like motion [ 3032 ] . the stimulation sequences are controlled by a microcomputer and are manually initiated by the user , and a walker or crutches are used for weight and balance support . forward movement is predominantly achieved by sliding the walker in the double support phase or with the help of the crutches . the walking fes systems can be used in combination with mechanical braces to reduce the number of muscles to be controlled and to provide additional joint stability . , faiborn , oh ) is an fda approved surface fes system composed of a six - channel , microprocessor - controlled stimulator , skin electrodes , and a modified walking frame . this system has been evaluated for ambulation and other beneficial medical effects in several studies [ 3437 ] and is widely available . research - based implantable multi - channel fes devices for the restoration of lower limb movement in motor - complete sci subjects have been developed by several research groups . the feasibility of the application of implants utilizing either epimysial electrodes or cuff electrodes for the generation of standing and some stepping movements was tested in a few sci subjects [ 3840 ] . generally , the walking fes systems for motor - complete sci subjects are not a substitute for a wheelchair , but used as an active means of exercise they provide many benefits including improved blood flow to the lower limbs , increased muscle mass and strength , reduced spasticity , improvement in skin trophic , and cardiovasular benefits . in people with motor - incomplete sci , timed peroneal nerve stimulation can be used to facilitate the swing phase during locomotor training on a treadmill to augment gait rehabilitation in the early phase after injury . candidates for chronic use of peroneal electrical stimulators are sci individuals , who have sufficient trunk and knee extensor function but weak flexor activity of the lower limbs . in the chronic phase of incomplete sci , the diversity of injury profiles and residual motor function results in a broad variety of impaired gait patterns . by supplementing the individual residual motor control , such applications can exploit all of the characteristic advantages of fes to either achieve local or more widespread synergistic responses by stimulation of the appropriate peripheral nerves , to produce immediate muscle force by efferent stimulation or to utilize central mechanisms following the sensory input produced either directly or secondary to the evoked movements . epidural scs is generally associated with the treatment of chronic neurogenic pain conditions and in fact , scs for pain control is the most common modality of all neuromodulation therapies . yet , scs can be an effective method in the alleviation of various disorders of the motor system . the application of scs in motor disorders resulted from observations made by cook and weinstein when treating an ms patient for pain . cook 's work was repeated and the finding of improved motor control and spinal spasticity in patients with ms was confirmed [ 4648 ] . illis and colleagues observed immediate and profound improvements of motor , sensory and bladder function in 2 patients with ms treated by temporary epidural scs . the first patient with signs of an upper motor neuron lesion with spasticity regained unaided walking capability with scs . electrodes were positioned between the c3/c4 and t1/t2 vertebral levels and intermittent stimulation was applied at a frequency between 50 hz and 100 hz . such stimulation elicited paraesthesiae in the cervical or thoracic dermatomes corresponding to the segmental level of the epidural electrodes and , with increasing stimulus intensities , in both lower limbs or in all four extremities . in all cases , a striking improvement of the spasticity of the lower ( and the upper ) limbs was confirmed by neurophysiological evaluation , with the best results observed in the ms patients . in subsequent studies of separate groups with increasing numbers of individuals treated it became clear that scs augmented motor function only in a portion of the tested patient populations with inter - individually varying improvements . davis et al . reported improvements of ambulation with increased endurance and muscle strength in 44 of 69 patients with ms . found significant improvement in 5 of 18 ms patients with mobility problems treated with scs . the beneficial responses included a feeling of lightness of the legs , the ability to stand and walk more easily , increased endurance , and regained unaided walking capability . the stimulation was carried out at 33 hz and the electrodes were placed between c6/c7 and t9/t10 vertebral levels . stimulation which produced improvements had to be adjusted to generate bilateral sensation into the legs ( by adjusting the medio - lateral electrode position and the stimulus intensity ) . the variability of the effects of scs is also reflected by a later work of the same group suggesting that scs did not have a major quantifiable impact upon motor disability in ms . the effect of scs in a large number of patients with various motor disorders , including 130 ms patients treated with scs , was discussed by waltz . among the ms patients , spasticity was abolished or significantly decreased in 58% , with increased ease in carrying out voluntary motor function . weakness improved in 33% , most markedly in the hip flexors , with a positive impact on gait . electrodes were placed between the vertebral levels c2 and c4 and frequencies of 1001500 hz were applied , with most patients responding best to 100200 hz . electrophysiological studies of the effects of scs during the treatment of ms , including recordings of cervical somatosensory and brainstem evoked potentials , indicated that scs acted at spinal cord and brainstem levels . the detected changes in the evoked potentials in association with scs were interpreted as variations of the efficacy of afferent volleys in engaging neural circuits , as in the dorsal horn of the spinal cord and the nucleus cuneatus . gybels and van roost suggested in their review of numerous studies of scs in motor disorders that the working mechanisms of scs are beyond local effects and rather due to an increased descending inhibition of excessive spinal reflex activity . illis hypothesized that scs might work through modulation of the central excitatory state and neurotransmitter release and of the afferent inflow . the variety of rostro - caudal sites of scs in the studies reviewed above , ranging from c2 to t10 vertebral levels , indeed suggests the activation of a rather diffuse system involving ascending and descending tracts ( the latter by synaptic activation ) and segmental circuits . this fact together with the pathophysiological complexity of ms might have contributed to the variety of results reported . our understanding is that the therapeutic specificity of scs hinges on the direct involvement of specific segmental spinal circuits through the precise placement of the implanted electrodes . with regard to lower limb functions , considerable progress has been made in recent years in recognizing the remarkable capabilities of the lumbar spinal cord circuitry ( located at t11t12 vertebral levels ) to control motor function and how they can be utilized by lumbar scs [ 5660 ] . studies on the effect of scs targeting these circuits involved in the control of muscle tone and lower limb movement will be presented in the following sections . richardson , mclone and colleagues described the alleviation of severe spasticity and flexor spasms in 6 individuals with post - traumatic , complete thoracic sci by scs in a series of case reports . epidural electrodes were placed below the injury between the l1 and l4 vertebral levels , and the effective stimulation frequencies were 3375 hz . did not confirm a satisfactory effect of scs on spasticity in 15 chronic sci patients , with epidural electrodes placed rostral to the lesion level ( see personal communication of dimitrijevic in ) . barolat and colleagues , on the other hand , concluded in a larger series of cases that epidural scs is effective and safe in the management of spasticity and spasms in sci individuals . waltz found improved motor function in 65% of 303 sci patients treated by cervical scs . benefits included decreased spasticity and spasms and augmented function in muscle groups retaining some voluntary movement . dimitrijevic and co - workers investigated the efficacy of scs for spasticity control in 59 sci patients and obtained marked or moderate effects in 63% of the patients . the epidural electrodes were placed at c2 to t12 vertebral levels and continuous stimulation was applied at 3050 hz . the variability of the efficacy of stimulation did not depend on the severity of spasticity , but on the rostro - caudal electrode site . considering their own results and the reports of richardson et al . and siegfried et al . , they concluded that the optimal position for epidural leads in patients with thoracic sci was below the level of the lesion . it was suggested that such thoraco - lumbar scs would lead to antidromic activation of residual , longitudinal structures of the spinal cord below the injury with resulting modulation of segmental spinal circuits activity involved in the generation of spasticity . in incomplete sci patients with partial functional preservation of ascending and descending neural tracts , long loop dorsal column spinal mechanisms modulating the excitability of segmental circuits were suggested as additional mechanism . in view of the motor control capabilities of the upper lumbar spinal cord circuitry ( see also below ) , pinter and colleagues revisited the use of scs in chronic sci individuals with severe lower limb spasticity . rather than just placing the epidural lead at some level below the sci , they precisely positioned the electrodes at the segmental levels of the upper lumbar spinal cord . the positioning of the lead was controlled under fluoroscopy and guided by intraoperative elicitation of muscle twitch responses ( see and cf . posterior root - muscle reflexes , below ) . based on the segmental muscle innervations , the target position required the lowest thresholds for responses evoked in the adductors and quadriceps . eight subjects with chronic , post - traumatic sci and severe lower limb spasticity were studied . lesions were at c5c6 ( n = 3 ) and t3t6 ( n = 5 ) levels and were classified as grade a ( n = 5 ) , b ( n = 2 ) and c ( n = 1 ) according to the american spinal injury association impairment scale ( ais ) , respectively . the neurophysiological assessment showed a significant suppression of lower limb spasticity in all subjects , when the lead was over the target site and the stimulation frequency in a range of 50100 hz . furthermore , the same study demonstrated that there was no modification of lower limb spasticity when the epidural contacts were located over the lowest thoracic spinal cord segments . at this more rostral stimulation site , the specificity of the segmental position of the stimulating epidural contact strongly suggested that the inhibitory mechanism were due to the stimulation of segmental afferents within the closest posterior roots that in turn trans - synaptically activated local neural processors in the lumbar spinal cord . note , that the posterior roots contain the whole range of sensory fibers , including group i afferent fibers that arise in muscles , whereas there are differences in the intramedullary projections of group i fibers and group ii cutaneous afferent fibers within the posterior columns . the control of lower limb spasticity by epidural stimulation of the upper lumbar spinal cord requires individual identification of effective stimulation parameters , involving stimulation through different contacts along the epidural lead ( fig . during such testing in the clinical practice of scs it became obvious that the stimulation did not only attenuate spinal cord activity , but also produced a variety of non - patterned and patterned motor outputs to the lower limb muscles , depending on the stimulation parameters . dimitrijevic and colleagues reported in chronic , motor complete sci subjects that lumbar scs could produce rhythmic activities in the paralyzed lower limbs . an example of locomotor - like , alternating electromyographic ( emg ) activity in groups of antagonistic muscles generated by lumbar scs is shown in fig . the functional separation of the lumbar spinal cord from supraspinal structures was demonstrated in these subjects by clinical and neurophysiological assessment . the influence of proprioceptive feedback in the rhythm - generation of the muscle activity could be assumed to be insignificant , since all subjects were studied in a supine position that limited lower limb loading and hip extension . it was thus demonstrated that the human lumbar spinal cord contains neural networks that can generate coordinated oscillating activity in response to tonic neural input signals , even after chronic disconnection from supraspinal structures . for the generation of rhythmic motor outputs to the lower limbs , scs frequency had to be within the range of 2560 hz . subsequent studies repeated and verified these results [ 7274 ] . in addition , it was found that non - patterned electrical stimulation of the lumbar spinal cord at 515 hz could generate and retain bilateral lower - limb extension in motor complete sci subjects in supine position . when the subjects lower extremities were manually moved to a position of hip and knee flexion , and scs was subsequently applied at 515 hz , a brisk and strong extension movement of the lower limbs was produced . the associated emg activities from the lower limb muscles revealed characteristic and reproducible temporal amplitude modulations ( fig . when the position of full extension was reached , and stimulation was continued , the limbs further remained in this position , with the muscles clearly contracting . in the same subjects , the extension - like muscle activities were replaced by rhythmic ones when the scs frequency was increased to 30 hz , without changing the site or intensity of stimulation . electrophysiological and computational studies revealed that the structures directly , electrically stimulated by epidural lumbar scs are predominantly afferent fibers of the posterior roots . the tonic neural drive to the lumbar spinal cord via the posterior root afferents was suggested to produce parallel effects . the volleys elicit segmental muscle responses in the lower limbs , so - called posterior root - muscle reflexes , and co - activate lumbar interneuronal circuits via synaptic projections . their integration into the generation of motor outputs most probably relies on temporal and spatial summation processes of the postsynaptic potentials produced . despite the above discussed findings and the clear potential for clinical application , few human studies have capitalized on the intrinsic motor control mechanisms of the lumbar spinal cord that remain below a spinal cord lesion . in 2 cases of chronic , motor complete sci individuals , the rhythmic emg activity produced by partial body weight - supported , manually assisted treadmill - stepping was immediately augmented when epidural lumbar scs at 2050 hz was supplied ( fig . it was thus shown that the rhythmic sequences of proprioceptive feedback associated with the mechanical events during passive stepping and the tonic neural drive produced by scs could be integrated by the lumbar spinal cord in the generation of motor output . furthermore , rhythmic activity could be produced in muscles that did not respond to manually assisted treadmill - stepping alone , and the timing of the activity relative to the gait cycle could be shifted by the stimulation . a similar augmentation of rhythmic muscle activities on a moving treadmill belt by scs was recently demonstrated in a case study of a subject with a clinically motor complete and sensory incomplete sci , but this subject had undergone an extensive locomotor training . rhythmic emg activity was generated when epidural lumbar scs at 3040 hz and step - related proprioceptive feedback were provided . independent gait - like movements were not achieved in this study either . yet , scs could generate full weight - bearing standing without manual facilitation when the stimulation frequency was set at 15 hz . additionally , 7 months after implantation , epidural scs revealed residual voluntary control of some lower limb movements that was only present when stimulation was supplied ( see also ) . in 2 wheelchair - dependent individuals with motor and sensory incomplete sci classified as ais c , herman and his team investigated a combined therapy of locomotor training and scs [ 8083 ] . they applied locomotor training until the participant reached a plateau in treadmill gait performance and subsequently employed epidural scs in conjunction with locomotor training . the initial locomotor training without scs improved the subjects ability to step on the treadmill , however , the improvements did not translate successfully into over - ground ambulation . acute effects of scs when applied during treadmill stepping included improved muscle activation patterns and an enhanced level of emg activity . after the period of combined training , both subjects were able to transfer the coordinated movements acquired through locomotor training with scs into functional over - ground ambulation . in both subjects , 2 epidural leads were placed over the thoraco - lumbar spinal cord in parallel , each located few millimeters off the physiological midline on either side , and stimulation frequency was between 20 and 60 hz . the thoraco - lumbar stimulation might have accessed the lumbar locomotor circuits by antidromic activity in afferent branches within the posterior columns of the spinal cord white matter or by stimulation of the respective segmental posterior roots due to current spread . , there is clear evidence for the presence of a remarkable motor control capacity within the human lumbar spinal cord that remains operational even after chronic separation from supraspinal structures . this remaining functional capacity can be set into operation by non - patterned stimulation delivered via intact segmental input pathways . in spite of being provided through afferent projections , such tonic activity is characteristic for descending neural drive rather than for proprioceptive feedback . frequencies of 515 hz promote extension , stimulation at 2560 hz can generate or facilitate rhythmic activity of the lower limbs , and 50100 hz can attenuate exaggerated spinal activity affecting the lower extremities . epidural lumbar scs does not activate spinal cord circuitries directly , but trans - synaptically through the electrical stimulation of posterior root and spinal cord afferents . posterior root fibers of the l2 to s2 spinal cord segments can be consistently stimulated by transcutaneous scs with skin electrodes [ 8688 ] . the technique uses stimulating electrodes over the paravertebral skin between the t11 and t12 spinous processes , and large reference electrodes over the lower abdomen ( fig . the biophysics leading to the specific , localized depolarization of posterior root fibers in spite of the distant stimulation is well - understood . the ligaments between the spinous processes and laminae and the intervertebral discs between the vertebral bodies reduce the transversal electrical resistance of the thoraco - lumbar spine and allow some current produced by transcutaneous scs to flow through the vertebral canal . within the vertebral canal there , the excitation threshold of the posterior root fibers is considerably reduced by non - uniformities of the anatomy along the fiber paths and changes of the fiber path direction with respect to the generated field . potential therapeutic applications of tonic transcutaneous scs for the alleviation of spinal spasticity and the enhancement of neural control of locomotion after sci are currently being investigated . in a pilot study , it was explored whether lumbar transcutaneous scs could temporarily modify spinal spasticity in 3 incomplete sci subjects classified as ais d . stimulation was applied in a supine position for 30 min at 50 hz and an intensity producing paraesthesiae in lower limbs . before and immediately after stimulation , the subjects spasticity and residual voluntary motor control were assessed clinically and neurophysiologically . in the post - stimulation evaluation , emg associated with achilles clonus , passive knee movement and mechanical plantar stimulation were reduced , and volitional rhythmic ankle movement was improved . two subjects ( one using a walker , the other one 2 crutches ) considerably increased their walking speed after scs . early exploratory studies have shown that tonic transcutaneous lumbar scs applied during treadmill stepping generated or modulated rhythmic lower limb emg activities in motor complete and incomplete sci . in the subjects with motor complete sci , manually assisted and body - weight supported treadmill stepping ( without stimulation ) produced only low - amplitude emg activity in the paralyzed lower limbs . when tonic transcutaneous scs at 30 hz was additionally supplied , rhythmic patterns of extensive emg amplitudes could be produced ( fig . 2b and c ) . that the induced rhythmic activities were largely caused by the tonic transcutaneous scs was reflected by the fact that the emg consisted of series of stimulus - triggered posterior root - muscle reflexes . while the first results of the combined application of transcutaneous scs and assisted treadmill stepping were very encouraging in motor complete sci subjects , independent functional gait - like movements were not yet produced . remarkable effects were achieved in 4 individuals classified as ais d. continuous transcutaneous scs at 30 hz and a sub - motor threshold level producing paraesthesiae in the lower limb dermatomes immediately modified the voluntarily generated lower limb activities in a gait phase appropriate way . the consciously controlled , yet augmented muscle activity led to changes of the stepping kinematics as well , including increased ranges of hip and knee movement and stride lengths . transcutaneous scs combines the characteristics of fes and neuromodulation techniques and has all advantages of a non - invasive method . using skin electrodes , it can be applied for repetitive interventions rather than for chronic stimulation therapies . studies in progress will clarify the role of transcutaneous scs in restorative neurology , particularly its efficacy in enhancing remaining motor capacity in people with various upper motor neuron dysfunctions . numerous research efforts are being conducted for the biological repair of the damaged cns that focus on the lesion site and the interrupted neural pathways . even after severe cns damage , however , there remains a distal , potentially functional apparatus of spinal cord , peripheral nerves and muscles . in the present review we discussed fes and scs as restorative neurology interventions in motor disorders that utilize this residual system . fes can provide for the immediate control of motor functions and generate sufficient force to produce movement . scs modifies the steady - state of neural circuits operation and their response to physiological input signals a common principle of neuromodulation therapies . the concept of restorative neurology to augment surviving cns capabilities is not new [ 9597 ] . yet , it received new appreciation by the accumulated evidence for the existence of neural circuits within the human lumbar spinal cord that can execute complex stereotyped motor tasks in response to rather unspecific stimuli . being deprived of ( sufficient ) supraspinal drive , neural processing and pattern generating networks caudal to a spinal cord lesion lose an adequate , sustainable state of excitability to be fully operational . furthermore , the altered balance of excitation and inhibition contribute to the clinical pictures of spasticity . scs provides a multi - segmental tonic neural drive to these circuitries and tunes their physiological state to a more functional level . these concepts need to be followed by technological and clinical advancements for the benefit of those who suffer from cns damage . epidural electrode arrays covering the dorsal columns as well as the dorsal root entry zones of the lumbosacral spinal cord in combination with multi - channel pulse generators are needed to allow for the spatially selective stimulation ( of multiple targets ) using field - steering methods . to mimic altered or absent supraspinal drive , stimulation modes with more flexible control of time parameters ( e.g. modulated stimulation frequencies ) need to be tested for the generation of locomotor activity . specific adaptations could build on such basic walking output by the combined utilization of scs with methods of fes or intraspinal microstimulation . scs in combination with repetitive , task - specific training can have a major impact on the functional recovery in patients with severe damage to their motor system . the confirmation of this potential by clinical trials with statistically appropriate subject populations or by alternative objective approaches will be one of the future challenges . such clinical programs will require the combined efforts of neurologists , human neurophysiologists , therapists and biomedical engineers at centers where comprehensive evaluation and long - term follow - up can be carried out .

in medical dictionaries , the word periodontium comes from the greek terms peri- , which means around , and -odons , which means tooth . those tissues are gingiva , cementum , periodontal ligaments , and alveolar bone [ 1 , 2 ] . a long time ago , it has been found that periodontium of the primary dentition differs from that of the permanent dentition in several aspects . the gingiva in primary dentition appears to be more reddish , vascular , and flabby and to lack stippling [ 1 , 4 ] . and the periodontal ligaments in children are wider and have less dense fibers [ 1 , 3 , 4 ] . the alveolar bone in primary dentition has less trabecula and calcification , more marrow spaces , and greater blood supply and lymphatic drainage [ 1 , 3 , 4 ] . at the molecular level , some investigators reported that periodontium of the primary dentition resorbed more easily because it contains more sialoprotein and osteoprotein , which facilitate the binding of odontoclast [ 1 , 57 ] . periodontal diseases constitute a group of conditions that are considered nowadays ubiquitous among children , adolescents , and adults . the term periodontal diseases includes any inherited or acquired disorders of the tissues that are investing and supporting the teeth ( gingiva , cementum , pdl , and alveolar bone ) . another researcher defined periodontal diseases as chronic infectious disorders caused primarily by bacteria . in 1996 , albandar et al . assessed the prevalence of gingivitis among large group of adolescents in the united states and found that 82.1% of the participating subjects were having gingivitis . similar findings of high prevalence of gingivitis among children and adolescents were reported by other studies worldwide [ 16 , 17 ] . albandar et al . , in another study , assessed the prevalence of early - onset forms of periodontitis among group of us adolescents and reported that 0.6% of the subjects were having juvenile periodontitis at the age of 1315 , and 2.75% of the subjects were having chronic periodontitis at the age of 16 - 17 . low prevalence of periodontitis among children and adolescents was reported by other studies in different populations . many researchers have observed larger amount of plaque and less inflammation in relation to the amount of plaque in children compared to the adults [ 3 , 4 , 14 ] . furthermore , experts and clinicians noted that most of the periodontal diseases that affect children and adolescents are reversible and cause little tissue damage compared to the adults . periodontal diseases are most commonly caused by pathogenic microorganism in the oral biofilm or dental plaque that accumulated around the teeth due to poor oral hygiene [ 3 , 4 ] . the evidences indicate that periodontal diseases develop when the numbers of gram - negative bacteria and anaerobes in subgingival plaque increased [ 20 , 21 ] . numerous research efforts were implemented in order to identify bacterial species that are associated with the periodontal diseases [ 22 , 23 ] . the most common periodontal - diseases - associated microorganisms were aggregatibacter ( actinobacillus ) , porphyromonas gingivalis , tannerella forsythensis , and spirochaete treponema denticola [ 2 , 2428 ] . recent studies implicate fungi , such as candida albicans , and herpes viruses in the pathogenesis of periodontal diseases among immune - compromised children [ 2931 ] . however , genetic , developmental , traumatic , neoplastic , and metabolic factors contributed to the cause of these diseases [ 9 , 11 , 24 ] . furthermore , some systemic diseases and medications also have periodontal manifestations [ 24 ] . over the last few decades , regardless of the causative factors , periodontal diseases are divided into destructive and nondestructive form . gingivitis is a reversible and nondestructive form of periodontal diseases [ 14 , 32 , 33 ] . it is the inflammation of the marginal gingiva that may progress to include free and attached gingiva but causing no attachment loss [ 9 , 11 ] . based on clinical findings and diagnosis , gingivitis was subdivided into infectious and noninfectious forms as in figure 1 [ 14 , 3437 ] . on the other hand , the irreversible and destructive form of periodontal diseases is periodontitis . it is the inflammation of the tooth supporting tissue , which is accompanied by loss of connective tissue attachment and breakdown of the supporting alveolar bone [ 9 , 11 ] . periodontitis may progress to cause exposure of the roots , mobility , and premature loss of the teeth . in 1989 , the american academy of periodontology set criteria in order to distinguish various forms of periodontitis . those criteria are ( 1 ) age at onset , ( 2 ) distribution of the sites affected by the disease , ( 3 ) presence or absence of the systemic diseases , ( 4 ) rate of the disease progression , ( 5 ) response to treatment , and ( 6 ) presence or absence of specific host or microbial factors ( the consensus of the world workshop in clinical periodontics ) . the most recent classification of periodontal diseases was introduced in 1999 by international workshop of periodontology and includes greater variety of periodontal diseases categories [ 3 , 38 , 39 ] . however , this paper will not follow specific classification system but rather will focus primarily on the periodontal diseases that are most commonly seen in pediatric dental patients . as mentioned earlier in this paper , gingival problems , diagnosis of various types of gingivitis relied mainly on the clinical findings and manifestations [ 3 , 32 ] . those findings include redness and edema of the marginal gingiva and bleeding upon probing [ 24 ] . as disease persists , gingival margin may become rolled , interdental papilla may become enlarged and bulbous , bleeding may start spontaneously , and probing depth may increase as a consequence of gingival overgrowth ( hyperplasia and hypertrophy ) [ 2 , 3 , 32 , 34 ] . histologically , ulceration of the sulcular epithelium was observed both in children and in adolescents [ 32 , 34 ] . however , researchers have noted predominance of t - lymphocyte infiltrate in gingivitis in children compared to b - cell ( plasma cells ) infiltrate in gingivitis in adolescents ( ranney et al . , 1981 , and page and schroeder , 1976 ) [ 3 , 4 ] . although the microbiological picture of gingivitis in children and adolescents has not been completely characterized , certain bacterial species have been found in experimental studies . those species were aggregatibacter ( actinobacillus ) sp . , capnocytophaga sp . , leptotrichia sp . , and selenomonas sp . . some gingival inflammation normally accompanies eruption process [ 4 , 9 , 11 ] . poor oral hygiene by neglect or as a consequence of malalignment of the erupting teeth will aggravate gingival inflammation [ 24 , 9 , 11 ] . usually , the condition will subside as the oral hygiene improves and the tooth reaches normal occlusion [ 2 , 4 , 9 , 11 ] . pubertal gingivitiswhich isalso called steroid hormone - related gingivitis is defined as exacerbation of gingivitis by fluctuation in gonadotrophic hormone levels during puberty [ 3 , 4 , 11 ] . a similar condition is seen during pregnancy ( loe , 1965 ) and in females taking contraceptives ( kalkwarf , 1978 ) [ 24 , 11 ] . the phenomenon of this condition can be explained as any increase in the levels of estrogen and progesterone in the gingival tissues resulting in vasodilatation and proliferation , increase in gingival vascularity , and increase in susceptibility of inflammation in the presence of local factors [ 4 , 11 , 40 , 41 ] . pubertal gingivitis is characterized by swelling of the interdental papilla , with spontaneous gingival hemorrhage [ 3 , 4 ] . professional prophylaxis and removal of the local factors combined with good oral hygiene regimen at home will result in major improvements . in some cases mouth - breathing causes desiccation of the oral tissue and consequently gingival inflammation and halitosis [ 4 , 11 ] . immediate management for the problem includes ( 1 ) maintaining good oral hygiene , ( 2 ) lubrication of the tissue , and ( 3 ) the use of the oral screen to cover the tissue during sleeping . elimination of the problem requires comprehensive treatment plan by an orthodontist and an otolaryngologist [ 3 , 26 ] . certain classes of medications have been approved to cause gingival overgrowth and aggravate gingival inflammation in the presence of local factors [ 3 , 4 , 41 ] . those medications are cyclosporine ( immune - depressant ) , phenytoin ( anticonvulsant ) , and calcium channel blockers ( antihypertensive ) [ 2 , 3 , 11 , 41 ] . gingival overgrowth was noted in 30% of patients taking cyclosporine , 50% of patients using phenytoin , and 15% of patients medicated with calcium channel blockers such as nifedipine , verapamil , and amlodipine [ 3 , 41 , 42 ] . this kind of gingival overgrowth usually starts at the interdental area and then spreads to include marginal gingiva [ 3 , 4 , 41 ] . occasionally , it can be so severe to cover the incisal and occlusal surfaces of the teeth [ 11 , 41 ] . however , its severity is closely related to the amount of accumulated plaque [ 4 , 11 , 41 ] . however , the interaction between those drugs and/or metabolites and fibroblast will lead to fibroepithelial gingival overgrowth , epithelial acanthosis , increase in fibroblast number , and increase in collagen production . the management of this condition starts from improving patient 's oral hygiene by both mechanical and chemical plaque control techniques [ 4 , 11 ] . in addition , professional scaling and polishing are required to remove all the local aggravating factors [ 24 , 11 ] . sometimes , gingivectomy and gingivoplasty are needed for gingival recontouring in order to improve esthetic and hygiene [ 3 , 4 ] . however , a consultation with the patient 's physician can be done to determine the possibility of drug replacement [ 3 , 44 ] . there is strong evidence that hypovitaminoses and mineral deficiency associated with specific manifestation in oral and perioral area may lead to periodontal diseases [ 2 , 11 ] . for example , vitamin c deficiency will cause scurvy , which is manifested as a decrease in the production and maintenance of collagen . oral scurvy is characterized by painful gingival swelling , gingival edema , and hemorrhage on slight provocation [ 11 , 45 ] . results when severe vitamin c deficiency is combined with poor oral hygiene [ 11 , 46 , 47 ] . however , it is characterized by ulcerative gingivitis , fetid odor , rapid development of periodontal pocket , and tooth loss [ 46 , 47 ] . trench mouth or vincent 's infection is an acute gingival inflammation caused mainly by a special bacterial species called borrelia vincentii [ 4 , 9 , 11 ] . the risk factors include poor oral hygiene , stress , decreased host resistance , and hiv infection [ 4 , 11 ] . anug is characterized by punched - out interdental papilla that is covered with a grayish - white pseudomembrane , which may extend to cover marginal gingiva [ 4 , 48 , 49 ] . patients are usually suffering from strong continuous pain and fetid odor as a result of bacterial reaction 's end products , bacterial toxins , and tissue necrosis [ 4 , 49 ] . generalized systemic manifestation including low - grade fever , lymphadenopathy , and malaise is often accompanying anug [ 48 , 49 ] . both local and systemic therapy are needed for the treatment of anug [ 4 , 48 , 49 ] . the first step is professional gentle scaling to remove local deposits as well as necrotic tissue . patients are instructed to follow strict daily oral hygiene regimen [ 4 , 49 ] . oxidizing mouthwash such as chlorhexidine may help to restore microbial balance [ 4 , 48 , 49 ] . 250500 mg per dose of penicillin or erythromycin was recommended for five days [ 4 , 48 , 49 ] . flagyl ( metronidazole ) is approved by evidence to help in eliminating the acute symptoms rapidly . primary herpetic gingivostomatitis is defined as an acute gingival condition that is caused by herpes simplex virus type i [ 3 , 4 ] . its clinical picture is characterized by a painful gingival inflammation and vesicles that are formed mainly on the dorsum of the tongue , hard palate , and gingiva . those vesicles ruptured eventually , leaving a painful ulcer with a yellow gray floor and red halo [ 3 , 4 , 11 ] . lymphadenopathy , fever , and malaise are common systemic features for herpetic gingivostomatitis [ 48 , 49 ] . it is commonly affecting children under the age of ten with a peak incidence at 24 years of age . the condition is self - limiting and required symptomatic treatment only [ 3 , 4 ] . although this form of periodontitis is considered more prevalent in adults , it can be seen occasionally in children and adolescents . comparing to aggressive periodontitis , chronic periodontitis is characterized by a low to moderate rate of progression that may include episodes of rapid destruction [ 9 , 11 ] . it is subdivided according to the percentage of the involved sites into localized ( < 30% ) and generalized ( > 30% ) [ 2 , 3 , 9 ] . furthermore , it can be subdivided according to the severity of the disease into mild ( 1 - 2 cal ) , moderate ( 3 - 4 cal ) , and severe ( 5 cal ) [ 9 , 11 ] . aggressive periodontitis which is also called juvenile periodontitis is considered to be prevalent in children and adolescents during circumpubertal period [ 3 , 10 , 11 ] . it is characterized by rapid loss of connective tissue attachment and alveolar bone with familial aggregation . aggressive periodontitis can be subdivided into localized ( lagp ) and generalized form ( gagp ) . localized aggressive periodontitis patients have interproximal attachment loss on no more than two teeth other than first permanent molars and incisors [ 3 , 10 , 11 ] . at the microbiological level , up to date , no single species of microorganism has been found in all cases of lagp [ 9 , 50 ] . however , aggregatibacter ( actinobacillus ) sp . in combination with bacteroides - like sp . and eubacterium sp . it is well documented that lagp is associated with a variety of functional defects in neutrophils [ 10 , 55 ] . generalized aggressive periodontitis patients have interproximal attachment loss on at least three teeth that are not permanent first molars or incisors [ 3 , 10 ] . it is usually affecting the entire dentition and is considered as a disease of adolescents and young adults . at the microbiological level , porphyromonas gingivalis and treponema denticola were isolated from most gagp cases [ 3 , 10 ] . patients with gagp have defective neutrophil functions and reduction in gp-110 [ 10 , 56 ] . furthermore , alteration in igg was reported to be present in both forms of aggressive periodontitis . the successful treatment of aggressive periodontitis includes surgical or nonsurgical periodontal therapy in combination with systemic antibiotic therapy [ 10 , 57 ] . according to the number of studies , the most successful antibiotic in the treatment of aggressive periodontitis is tetracycline alone or with metronidazole [ 10 , 5759 ] , followed by metronidazole in combination with amoxicillin in the presence of tetracycline resistance [ 10 , 60 ] . include a group of rare diseases that predispose the affected individual to highly destructive periodontal infections [ 8 , 9 , 12 ] . the most common systemic diseases and genetic disorders that are associated with periodontal conditions are listed in table 1 . the treatment of periodontitis as a manifestation of systemic diseases includes a combination of surgical and nonsurgical therapy in addition to antibiotic therapy [ 8 , 9 , 61 , 62 ] . however , the success of treatment of periodontitis as a manifestation of systemic diseases is considered unpredictable [ 8 , 9 ] .

amc contributed to the writing of the complete paper including , literature search , review of papers , drafting the manuscript , and major revision . not commissioned ; peer - reviewed by mohammed rashed amc contributed to the writing of the complete paper including , literature search , review of papers , drafting the manuscript , and major revision .

the main goal of endodontic treatment is to prevent and , when required , to cure apical periodontitis and maintain or reestablish periapical tissue health . to accomplish this objective , it is mandatory to control the microbial load inside the root canal system . the chances of a favourable outcome with endodontic treatment are significantly higher if infection is eradicated effectively by chemomechanical preparation before the root canal is obturated . however , if positive cultures can be obtained from the root canal at the time of root filling , there is a higher risk of treatment failure . in an attempt to improve disinfection , an interappointment dressing has been advocated to diminish the percentage of root canals with no cultivable microorganisms in comparison to those only treated with chemomechanical preparation . nevertheless , the two - visit treatment protocol did not improve the overall antimicrobial efficacy of the treatment . indeed , in all cases where viable microorganisms remain in the root canal system , the prognosis for repair is adversely affected [ 2 , 3 ] . presence of a smear layer after instrumentation reduces effectiveness of irrigants and temporary dressings in disinfecting dentinal tubules . moreover , complexity of anatomy translated into root canal system with its isthmuses , ramifications , and fins turns complete elimination of bacteria using instrumentation and irrigation into an almost impossible task . besides , bacteria persisting in biofilms show diverse phenotypes when compared with planktonic cells , including increased resistance to antimicrobial agents . it has been assessed that bacteria in biofilms are approximately 1000-fold less susceptible to effects of commonly used antimicrobial agents than their planktonic equivalents and are highly unaffected with phagocytosis by immune system . there are several mechanisms used by bacteria which allow them to adapt to the environment . biofilm formation , stress response , physiological adaptation , and the beginning of subpopulations of cells are among some of the adaptive mechanisms used by bacteria along with various systems involving the exchange of genetic material between bacteria . these mechanisms can support bacterial survival under the limiting environments , such as that found in the root canal . one of the most relevant features of adaptation for oral bacteria is the adhesion to surfaces leading to the formation of plaque biofilms , which not only serves to aid in their retention but also results in increased survival rate . biofilms form when planktonic bacteria in a natural liquid phase are deposited on a surface containing an organic conditioning polymeric matrix or conditioning film . in this dynamic process , several organisms coadhere to the surface and grow with certain cells detaching from the biofilm over time . biofilm formation in root canals , as postulated by svensater and bergenholtz , is probably initiated at the moment of the first invasion of the pulp chamber by planktonic oral organisms after some tissue breakdown . biofilm disruption and disinfection of root canals are the most critical steps during treatment of an infected root canal system , which are essential to avoid persistence of microbial infection and achieve endodontic success . the mode of action and efficacy of a wide variety of cleaning , antimicrobial , and disinfecting agents such as naocl , chlorhexidine , ethylenediamine tetraacetic acid ( edta ) , citric acid , hydrogen peroxide , halogens , and ozone have been investigated [ 1518 ] . disinfecting agents and antimicrobial medicinal products routinely used in endodontics can be inactivated by dentin , tissue fluids , and organic matter [ 6 , 19 ] . moreover , some microbial species , such as enterococcus faecalis [ 20 , 21 ] and candida albicans [ 22 , 23 ] , show resistance to those agents and their efficacy is dependent on the concentration achieved and time of contact . most of these disinfectants with effective bactericidal activity are used at subtoxic level , but also at concentrations where toxicity is becoming a significant factor . searching for new methods to provide extra disinfection for root canal system without cytotoxic effects and to improve treatment outcome , innovative techniques including various laser wavelengths , hydraulic , sonic , and ultrasonic irrigation [ 2729 ] , nanoparticles , inactivation of efflux pumps , and photodynamic therapy ( pdt ) has been proposed in literature . pdt was discovered by chance at the very beginning of the twentieth century , when a combination of nontoxic dyes exposed to visible light resulted in microorganism cell death . as reviewed by henderson and dougherty in 1992 , oscar raab , a medical student working with professor herman von tappeiner in munich , introduced the concept of microbial cell death induced by interaction of light and chemicals . during the course of raab 's study , he demonstrated that the combination of light and dyes was much more effective in killing the microorganism paramecium . succeeding work in this laboratory coined the term photodynamic action and demonstrated presence of oxygen as an essential requisite for photosensitization to occur . years later , dougherty and coworkers clinically tested pdt in cutaneous / subcutaneous malignant tumours . combined effect of three elements , light , ps , and oxygen , has been termed photodynamic antimicrobial chemotherapy by wainwright and also recognized as antimicrobial photodynamic therapy and photoactivated disinfection . pdt uses a nontoxic dye , known as photosensitizer ( ps ) , on a target tissue , which is consequently irradiated with a suitable visible light of the appropriate wavelength to excite the ps molecule to the singlet state in presence of oxygen to produce reactive oxygen species ( ros ) . when ps absorbs light , this excited state may then undergo intersystem crossing to the slightly lower energy , but the longer lived , triple state can undergo two kinds of pathways known as type i ( reacting with the substrate ) and type ii ( reacting with molecular oxygen ) photoprocesses . the type 1 radical and reactive oxygen species pathway comprises an electron transfer step between the triplet ps and a substrate with generation of radical species . the finalist is then intercepted by ground state molecular oxygen yielding a variety of oxidized products . the baseline ps has two electrons in opposite spins ( singlet state ) in the low energy molecular orbital . subsequent to the absorption of light , one of these electrons is boosted into a high - energy orbital but keeps its spin ( first excited singlet state ) . this is a short - lived time species , nanoseconds , and can lose its energy by emitting light ( fluorescence ) or by internal conversion into heat . type 1 pathway frequently involves initial production of superoxide anion by electron transfer from the triplet ps to molecular oxygen ( monovalent reduction ) initiating radical - induced damage in biomolecules . superoxide is not particularly reactive in biological systems and does not by itself cause much oxidative damage but can react with itself to produce hydrogen peroxide and oxygen , a reaction known as dismutation that can be catalyzed by the enzyme superoxide dismutase ( sod ) . the way of the electron relocation between the ps and the substrate is controlled by the relative redox potentials of the two species . type 2 pathway , singlet oxygen , involves an electronic energy transfer process from the triplet ps to a receptor , most frequently oxygen , which is a triplet in its ground state . the final compound is converted to a highly reactive species , the singlet oxygen ( o2 ) . the excited singlet state ps may also undergo the process known as intersystem crossing whereby the spin of the excited electron inverts to form the relatively long - lived , in terms of microseconds , excited triplet state that has parallel electron spins . the long lifetime of the ps triplet state is explained by the fact that the loss of energy by emission of light ( phosphorescence ) is a spin forbidden process , as the ps would move directly from a triplet to a singlet state . photosensitized processes of types 1 and 2 depend on the initial involvement of radical intermediates that are subsequently scavenged by oxygen or the generation of the highly cytotoxic singlet oxygen ( o2 ) by energy transfer from the photoexcited sensitizer . it is difficult to determine without doubt which of these two mechanisms is more prevalent ; both types of reactions can happen simultaneously and the ratio between them depends on three singular features : oxygen , substrate concentration , and ps type . hamblin and hasan in 2004 stated that antimicrobial ps can be divided into three categories : ( i ) those that strongly bind and penetrate the microorganisms ( chlorin e6 ) , ( ii ) those that bind weakly as toluidine blue ( tb ) and methylene blue ( mb ) , and ( iii ) those that do not demonstrate binding at all such as rose bengal ( rb ) . understanding these mechanisms of action is essential because , in bacterial cells , outer membrane damage plays an imperative role , differently from mammalian cells , where the main targets for pdt are lysosomes , mitochondria , and plasma membranes . typically , neutral anionic or cationic ps molecules could powerfully destroy gram - positive bacteria , whereas only cationic ps or strategies which attack the gram - negative permeability barrier in combination with noncationic ps are able to kill multiple logs of gram - negative species . this difference in susceptibility between species in the two bacterial types is explained by their cell wall physiology . to understand better the pdt effect in those microorganisms , it is very important to analyse in detail the microbial cell walls . in gram - positive bacteria , the cytoplasmic membrane is surround by a relatively porous peptidoglycan layer and lipoteichoic acid that allows the ps to cross . different from this , the gram - negative bacteria cell envelope consists of an inner and an outer membrane which are separated by a peptidoglycan layer . the outer membrane forms an effective permeability barrier between the cell and the environment and tends to restrict the binding and penetration of several ps . fungi are provided with a thick cell wall that includes beta glucan and chitin offering a permeability barrier . in terms of pdt efficacy , in fungal wall , it was described as having an intermediate behavior between gram - positive and gram - negative bacteria . on the basis of these considerations , it appears that gram - negative bacteria represent the most challenging targets for any form of antimicrobial treatment . the mechanism of action of basic polymer ps conjugates is thought to be that of self - promoted uptake pathway . in this method , cationic molecules first dislocate the divalent cations , such as calcium ( ca ) and magnesium ( mg ) , from their position on the outer membrane where they act as an anchor for the negatively charged lipopolysaccharides molecules [ 40 , 41 ] . the debilitated outer membrane becomes slightly more permeable and allows even more of the cationic ps to gain access thus steadily increasing the disorganizations of the permeability barrier increasing ps uptake with each additional binding . it is thought that host cells only gradually take up cationic molecules by the process of endocytosis , while their uptake into bacteria is relatively fast . further important observation that has been made about these cationic antimicrobial ps concerns their selectivity for microbial cells compared to host mammalian cells . the ps efficiency in generating ros within membranes is dependent on the intrinsic characteristics of the ps in aqueous solution as well as their partition in the membrane . the early attack of singlet oxygen in membranes lipids is by the specific reaction with double bonds to form allylic hydroperoxides ; the efficiency of this reaction is dependent on the lowest ionization potential of the olefins and also on the availability of allylic hydrogens . photodynamic lipid peroxidation is an oxidative degradation of cell membrane lipids , also known as photoperoxidation , and it has been related to several microbial cytotoxic effects , such as increased ion permeability , fluidity loss , inactivation of membrane proteins , and cross - linking , which disrupts the intracellular homeostasis . consequently , necrosis is induced as a cell death process . a probable explanation is that ps bound to the membrane and generates most of the singlet oxygen , o2 , involved in photoperoxidation highlighting the double selectivity ( light and ps cellular localization ) and the fact that it works in multiresistant strains and does not encourage resistance . pdt 's lethal action is based on photochemical production of ros and not thermal and cavitation effects , as is the case with high power laser therapy . one of several pdt 's advantages clinically is the absence of thermal side effects in periradicular tissues and this property of pdt aspect makes it highly effective in eradicating microorganisms such as bacteria , viruses , and fungi without causing damage of adjacent tissues due to overheating . in recent years , pdt has been applied in several areas , particularly in periodontology [ 4850 ] , in general dentistry and also in endodontic field as an adjunct of classical irrigation solutions in root canal disinfection [ 52 , 53 ] . these studies suggest pdt 's potential as a therapeutic weapon , which aims to support endodontic antimicrobial treatment , especially enhancing irrigation solutions effect . the purpose of this narrative comprehensive literature review is to answer the focused question , is antimicrobial pdt efficacy better than that of sodium hypochlorite 's in root canal treatment ? for this analysis of the literature , we selected and analysed 29 studies using antimicrobial pdt in endodontic field , highlighting methodologies used and their reported effectiveness and efficacy . for this comprehensive narrative literature review , eligibility criteria were ( i ) articles published in english language ; ( ii ) original papers ; ( iii ) experimental studies ( in vitro and ex vivo ) ; ( iv ) clinical studies ( in vivo ) ; and finally ( v ) scientific reports of pdt efficacy in root canal disinfection / asepsis . the exclusion criteria were ( i ) unpublished data , ( ii ) conference papers , ( iii ) historic reviews , ( iv ) letters to editor , and ( v ) papers due to pdt outcomes in other fields ( outside of endodontics ) . as a first step , the aim was to investigate the terms endodontic , photodynamic therapy , and briefly , we used pubmed to identify medical subject headings ( mesh ) terms corresponding to each term . nevertheless , mesh terms use is not common to all articles , making this search method infeasible . then , exhaustive automated searches of cochrane collaboration , evidence based dentistry ( ebd ) , journal of evidence - based dental practice ( jebdp ) , nhs evidence , and pubmed ( figure 1 ) were performed from january 2000 up to and including may 2015 using various combinations of the following key indexing terms : ( a ) endodontic photodynamic therapy ; ( b ) antimicrobial photodynamic therapy ; ( c ) photo - activated disinfection ; ( d ) light - activated disinfection ; ( e ) laser - assisted photosensitization ; ( f ) root canal disinfection ; and ( g ) endodontic lasers . titles and abstracts of all articles resulting from electronic search were screened independently and in duplicate by 2 reviewers . any disagreement between reviewers was solved via debate , although in specific cases of disagreement that were not resolved with discussion , opinion of a senior commentator was required . hand searching of reference lists of original and reviewed articles that were found to be relevant was also performed . in a second step , full - text copies of all remaining articles were obtained and further meticulous assessment was performed independently by each reviewer to determine whether or not they were eligible for this study based on the specific inclusion and exclusion criteria cited above and proven for agreement . quality evaluation of randomized clinical trials and observational studies was performed using strobe ( strengthening the reporting of observational studies in epidemiology ) and consort ( consolidated standards of reporting trials ) statement criteria , respectively . literature search provided 62 titles and abstracts ; from those , 29 studies concerned this theme : 16 were performed in in vitro conditions , 6 were in vivo studies , and the last 7 readings were ex vivo . from all 29 papers included in this review , 16 ( 55.2% ) were in vitro studies ( table 1 ) . in data processing , authors classified all studies in three categories : category i , in vitro ; category ii , in vivo ; and finally , category iii , ex vivo , to describe and clarify studies ' details . in category i , 16 in vitro studies , only 5 ( 31% ) [ 5761 ] reveal best antimicrobial pdt outcomes when compared with sodium hypochlorite ( naocl ) in range of 0.5 to 6% . only one study performed by nagayoshi et al . reveals equal results between pdt and naocl ; the remaining 10 ( 62.5% ) studies [ 6372 ] showed pdt outcomes unhelpful when compared with naocl as a classical irrigant solution , in concentration range of 0.5 to 6% . in category ii , 6 ( 21% ) papers [ 35 , 58 , 7376 ] were analysed ( table 2 ) . all were performed in the human dentition , five [ 35 , 58 , 73 , 74 , 76 ] were performed in permanent dentition , and only one was achieved in deciduous teeth by prabhakar et al . . all studies in category ii ( 100% ) presented that pdt efficacy overthrew ( 0.52.5% ) naocl . group [ 58 , 74 ] and juri et al . tested only permanent uniradicular human teeth ( incisors and canines ) as samples . . considered deciduous molars as a prerequisite for his study . finally , bonsor et al . [ 35 , 73 ] used not only uniradicular but also permanent multiradicular teeth . in terms of endodontic diagnosis , garcez et al . in his first study used patients with necrotic pulps and periapical lesion ; then , in 2010 , his group performed a second study to assess pdt efficacy in teeth with previous endodontic treatment , endodontic retreatment . juri et al . in 2014 evaluated pdt antimicrobial outcomes efficacy applied also in endodontic retreatment . both studies [ 74 , 76 ] revealed pdt outcomes near 100% effective . in category iii ( ex vivo ) , 7 ( 24% ) papers [ 5 , 7782 ] were analysed ( table 3 ) . based on this , 3 ( 43% ) studies [ 5 , 78 , 79 ] revealed superior pdt outcomes compared to 0.56% of naocl and in one study by xhevdet et al . group showed 2.5% naocl irrigation showed similar results to 5 min irradiation of pdt , 10 mg ml phenothiazine chloride as ps irradiated with 660 nm light source . considering all 29 publications , 14 of them ( 48% ) [ 5 , 34 , 35 , 5761 , 7376 , 78 , 79 ] showed best pdt antimicrobial outcome compared to ( 0.56% ) naocl used alone ; 2 ( 7% ) [ 62 , 81 ] papers reveal similar effects between them and the last 13 ( 45% ) [ 6372 , 77 , 80 , 82 ] studies revealed supremacy of sodium hypochlorite ( 0.56% ) . the present narrative literature review was based on hypothesis that antimicrobial pdt efficacy was better than sodium hypochlorite in root canal asepsis . considering all studies chronologically organized in table 4 , 48% ( 14 papers ) showed pdt is more efficient than naocl ( 0.56% concentration ) used alone and 7% ( 2 papers ) reveal similarity in antimicrobial outcome effects between them . on the other hand , 45% ( 13 studies ) of studies reveal supremacy of sodium hypochlorite . from all studies , it must be observed that 55.2% ( 16 studies ) were conducted at in vitro conditions , revealing preferential experimental phase where pdt remains in the last two decades . this must be taken into consideration , when comparing with clinical pdt studies , in which evidence reveals unanimous evidence supremacy of pdt over naocl . the 29 studies analysed for this review revealed assessment of antimicrobial pdt efficacy was done through several parameters , from microbiological evaluation ( classical analysis ) to recent advanced imaging approaches . at the beginning , bacteriological experimental in vitro studies presented results through colony - forming units ( cfu ) . this approach overcomes limitation of direct microscopic counting of bacterial cells , where all cells , dead and live , are counted ; cfu estimates only viable cells of each group , before and after treatment , in planktonic suspensions and biofilms . results are given as cfu / ml ( colony - forming units per millilitre ) for liquids . this approach was used in 24 studies ( 83% ) [ 34 , 5766 , 6872 , 7481 ] ; bonsor et al . [ 35 , 73 ] used bacterial load scores , instead of the usual cfu , to evaluate pdt antimicrobial efficacy in clinical studies . muhammad et al . in 2014 over an ex vivo study elected the same evaluation unit as in bonsor et al . studies , repeating bacterial score , complemented with microbiological identification . scanning electron microscopic ( sem ) in vitro investigations have demonstrated the penetration of bacteria up to 1000 m into dentinal tubules and hence it is very difficult for normal irrigants to penetrate till this depth . naocl can penetrate in a range of 60150 m into dentinal tubules and of nd : yag laser at a range of 400850 m . enterococcus faecalis is known to colonize dentinal tubules up to depth of 6001000 m and conventional irrigants can not penetrate more than 100 m . with sem , bumb et al . 's in vitro study revealed bacteria found till the depth of 980 m ( control group ) and in pdt group achieved a depth of 890900 m free from microorganisms , which revealed pdt as a promising root canal disinfection approach . sem is a remarkably versatile technique , which reproduces the exact morphology of structures , but as the main disadvantage of dehydration of the sample . it was used in 10 ( 34% ) studies [ 61 , 65 , 68 , 69 , 71 , 72 , 7982 ] and esem ( environmental scanning electron microscope ) which allows preservation of the sample before and after light irradiation was not used in any study . cslm was used only in one study of george and kishen showing capability of obtaining in - focus images from selected depths allowing three - dimensional reconstruction of topologically complex objects with a specific hardware analysis . the same study also evaluated dark toxicity ( detail described in photosensitizers subchapter ) and ros production . pdt antimicrobial killing can be mediated by type i and type ii reactions , although singlet oxygen is the predominant chemical entity causing cell death . analysis and quantification of singlet / reactive oxygen species detection seem to be an excellent methodology to quantify antimicrobial pdt outcomes . however , of all studies analysed , only george and kishen performed ros quantification and state that the increased photooxidation potential and singlet oxygen generation were thought to have collectively contributed towards the biofilm matrix disruption and bacterial inactivation . photosensitizers ( ps ) , which were preferentially located at the bacterial cytoplasmic membrane , have been found to be very potent photoantimicrobial agents . one important exception is represented by acridines , such as proflavine or acridine orange , which mostly interpolate with dna bases . highest modifications of cell functions and morphology , triggered by photodynamic inactivation , are typically due to damaged membranous domains . this pattern of photoinduced subcellular damage is in agreement with lack of mutagenic effects , as well as with absence of selection of photoresistant microbial strains even after several photosensitization treatments . methylene blue ( mb ) , a well - established ps , has been used in pdt for targeting endodontic bacteria since 2007 and remains as one of the most used ; but the first ps used in endodontic field was toluidine blue ( tbo ) . hydrophilicity of mb , along with its low molecular weight and positive charge , allows it to cross outer membrane of gram - negative bacteria through porin channels [ 33 , 86 ] . mb predominantly interacts with anionic macromolecule lipopolysaccharide , resulting in generation of mb dimers , which participate in the photosensitization process . from all studies evaluated , 12 ( 41% ) [ 35 , 6365 , 67 , 68 , 70 , 71 , 73 , 79 , 80 , 82 ] used tbo as ps , while 10 ( 34% ) [ 5 , 59 , 61 , 65 , 66 , 69 , 75 , 77 , 78 , 87 ] studies used mb . the best antimicrobial pdt results were achieved with tbo and mb as ps in the same concentration , 15 g ml [ 5 , 65 , 70 , 71 , 82 ] . all concentration variations are studied first in preliminary findings to obtain fluorescence characteristics in ultraviolet - visible absorption spectra on a diode - array spectrophotometer to understand absorption pattern and to establish final concentration . in designing criteria for definition of second generation ps , it is clearly desirable that ps has zero or very low cytotoxicity in total absence of light and this indicates antimicrobial pdt efficacy results strictly from combination between ps and light source . reviewing literature in this aspect , only one study from george and kishen had this aspect in mind . the period of intimate contact between ps and substrate without irradiation , known as preincubation time ( pit ) , diverges in terms of ps used . it is also important that pit is fixed in total absence of light , even natural light . the most used tbo pit was 60 seconds ( s ) [ 35 , 67 , 73 , 80 , 82 ] from a range of 30300 s ( mean = 95.5 s ) and mb pit most used was 300 s [ 5 , 66 , 75 , 78 ] from a range of 60600 s ( mean = 353.3 s ) . phototherapy describes use of light in treatment of disease ; photochemotherapy , on the other hand , involves a combination of administration of a photosensitizing agent followed by action of light on tissues in which the agent is located . pdt kills microorganisms by combined action of visible light and a photosensitizing dye . from all 29 studies evaluated , laser wavelength gap referred to in literature was between 380 and 910 nm ( mean = 650.8 nm ) , while most used light source was a diode laser of 660 nm [ 5 , 34 , 58 , 65 , 66 , 69 , 7477 , 79 , 81 ] wavelength . some orthodox photosensitizers have lost their proficiency because they needed specific light source for each one and combination between them triggers the costs . several examples can illustrate this aspect : azpaste ( 685 nm ) ; indocyanine green ( 805 nm ) ; eosin - y , and curcumin ( 380500 nm ) which make them , nowadays , outdated . in terms of commercial light sources , there are three diode lasers that authors would like to remark : denfotex of 635 nm ( savedent ; denfotex , inverkeithing , uk ) [ 64 , 90 , 91 ] , helbo of 660 nm ( helbo photodynamic systems , grieskirchen , austria ) , and fotosan emitting in the red spectrum with a power peak at 628 nm ( fotosan ; cms dental , copenhagen , denmark ) [ 67 , 68 , 71 ] . delivery of pdt treatment with denfotex , according to the manufacturer 's recommendations , includes tbo as ps at a concentration of 12.7 mg l , applied in 120 s as preincubation time ( pit ) ; followed by an irradiation time ( it ) of 150 s with a laser output power of 100 mw using the spherical tip . helbo system advocates helbo endo blue ps , a mb dye , at a concentration of 10 mg l fully covering the root canal with a pit of 180 s ; after this time , according to the manufacturer 's recommendations , excess ps dye should be removed and light source applied for an it of 120 s and an output power of 75 mw with an attached 2d spot probe helbo photodynamic systems . meire et al . in 2012 performed an in vitro study comparing denfotex with helbo . the same team reported that log reduction with helbo system was higher than with denfotex ; however , the best results were achieved with 2.5% naocl for 300 s. several differences between the two systems were described and might account for the distinctive reduction outcomes in viable cells . first , the ps dyes are chemically different ; secondly , helbo blue ps is much more concentrated than denfotex ps . thirdly , following the ps application and the recommended pit , the ps excess has to be removed with the helbo system , dried canal , but not with the two other systems : denfotex and fotosan , where fiber is inserted in the liquid [ 67 , 68 , 71 , 91 ] . in the three pdt systems , while the helbo systems 2d spot probe is designed for two - dimensional exposure , denfotex and fotosan tips emit in three dimensions and this has strong implications for energy densities at the target . it seems that there is also a clear reduction in light exposure as irradiation time ( it ) : denfotex ( 150 s ) , helbo ( 120 s ) , and fotosan ( 30 s ) [ 67 , 68 , 71 ] . fotosan uses only tbo as a fotosan ps , available in three types of viscosities ( low , medium , and high ) , all at the same concentration ( 100 g ml ) and the light source with an output power of 100 mw . fotosan was evaluated in 3 ( 10.3% ) studies [ 67 , 68 , 71 ] , curiously all conducted in in vitro conditions with fotosan protocol it of 30 s. poggio et al . tested 30 s and also 90 s of it and declared that with the longer light exposure , it results in an increased percentage of bacterial reduction for different groups of enterococcus faecalis , streptococcus mutans , and streptococcus sanguis strains . for this reason , this group admits that fotosan needs to be applied into canal for at least 90 s , because 30 s of irradiation showed lower performance when compared to pdt with it of 90 s , although the same group reveals that the best outcomes were achieved with pdt 30 s of it combined with 5% naocl . irradiation time ( it ) is an important issue to considerer and , in this parameter , pdt studies outcomes are very dissimilar with a range between 30 s [ 63 , 68 ] and 1800 s . considering the most used wavelength of 660 nm , preference irradiation time is in the range between 30 s and 1200 s ( mean = 223 s ) . the last aspect considered in laser literature is the need for an intracanal fiber tip to spread light into root dentinal walls as well as within biofilms . from all studies explored in vitro effectiveness of pdt with and without use of an intracanal optical fiber . nunes et al . concluded that , under experimental conditions , pdt was effective against e. faecalis , regardless of whether or not it is applied through an intracanal fiber . considering the use of intracanal fiber , only 4 ( 13.8% ) studies [ 63 , 70 , 75 , 80 ] were not performed with intracanal fiber ( table 5 ) . prabhakar et al . , in these particular conditions , revealed in a clinical study that antimicrobial pdt performance is better than 0.5% naocl . when pdt is implemented in planktonic suspensions established in multiwells , light source was applied 20 mm away from well . considering intracanal fiber , fiber tip diameter most used was 400 nm [ 59 , 62 , 64 , 72 , 77 ] . in terms of intracanal fiber location inside root , it varies from full working length ( wl ) [ 34 , 58 , 62 , 64 , 66 , 71 , 76 , 79 , 81 , 82 ] , the most prevalent , to wl-1 millimeters ( mm ) [ 57 , 74 ] , wl-2 mm [ 61 , 68 ] , wl-3 mm [ 6769 ] , and wl-4 mm [ 35 , 73 ] . contemplating the same device , intracanal fiber , in terms of applying movements to itself or inserting endodontic tip static inside root canal to improve the best light diffusion through root canal . the former was applied in 5 studies [ 34 , 58 , 65 , 66 , 79 ] with spiral movements from apical to cervical and latter maintained static [ 64 , 76 , 77 ] inside root canal orifice or at wl [ 64 , 76 ] . in literature , when pdt studies are accomplished in teeth , the majority of them are performed in human single rooted tooth specimens with no evidence of caries or defects and radicular pathology . considering tooth type , there is only one study performed in deciduous teeth ; the majority was achieved in permanent uniradicular human teeth . however , four studies used not only uniradicular but also multiradicular teeth [ 35 , 73 , 78 , 82 ] . besides , decayed teeth are also studied in deciduous and permanent teeth . slaughterhouse bovine teeth are convenient to use in antimicrobial pdt studies because of their match with human dentine ; more precisely , their dentinal tubules are very similar to human teeth in quantity , size , diameter , morphology , and density . moreover , bovine teeth are simple to acquire and reduced size makes handling easier ; in this term , they were used in 2 ( 7% ) studies [ 72 , 80 ] . only one study , performed by nagayoshi et al . , was executed in a resin block which attempts to mimic an in vitro model of apical periodontitis . in the most common experimental model , dental specimens are decoronated to a standard length of 12 mm [ 67 , 68 , 78 , 79 ] although gap value is very wide , from 8 [ 59 , 77 ] to 15 mm [ 66 , 81 ] or complete root canal length . patency of apical foramina is established and then mechanical [ 35 , 58 , 61 , 6368 , 70 , 71 , 73 , 74 , 76 , 78 , 79 , 81 , 82 ] instrumentation is performed using nickel - titanium rotary files , predominantly in a coronoapical ( crown - down ) technique [ 35 , 58 , 61 , 6368 , 70 , 71 , 73 , 74 , 76 , 78 , 79 , 81 , 82 ] from canal orifice to apical third , until it reaches the value of master apical file ( maf ) of k ( kerr ) file 40 [ 58 , 59 , 68 , 70 ] . however , other maf have been described , such as 35 [ 57 , 79 , 81 ] and 30 . in terms of irrigation with disinfecting agents , those are used for smear layer ( sl ) removal , lubrication , debris removal , and antimicrobial effects . sl is composed of organic and inorganic components like vital or necrotic pulp tissue , microorganisms , saliva , blood cells , and tooth structure . among irrigation solutions , sodium hypochlorite ( naocl ) is the classical irrigant most used in endodontic therapy as a powerful antibacterial organic tissue dissolving agent . naocl penetrates to a depth of approximately 130 m to 160 m into dentinal tubules whereas tubular infection may occur closer to cementum - dentin junction ( up to 1000 m ) . bumb et al . demonstrated in scanning electron microscope ( sem ) penetration up to 1000 m into dentinal tubules of e. faecalis and compared penetrating power between a high power laser ( nd : yag ) that can go to a range of 400850 m and pdt group that reaches as deep as 890900 m . considering naocl as an unquestionable irrigation solution , its universal effective minimal concentration remains unclear . apart from various outcomes reported by previous studies on comparative effectiveness of hypochlorite at different concentrations , it is regularly accepted that effectiveness of naocl is proportional to its concentration [ 24 , 72 , 94 ] . in antimicrobial pdt studies , naocl concentration range is between 0.5 [ 57 , 67 , 75 , 80 ] and 6% [ 68 , 78 ] and mainstream of studies used 2.5% naocl concentration [ 34 , 58 , 62 , 64 , 65 , 70 , 71 , 74 , 76 , 79 , 81 ] . due to the fact that naocl has an influence upon only organic components of sl , it should be used with demineralizing agents , which can remove inorganic component of smear layer . concerning sl elimination , only 3 readings [ 35 , 70 , 73 ] reported citric acid as a sl deletion , one at 10% and two at 20% from the same author , bonsor et al . but the most popular sl removal is by far 17% ethylenediamine tetraacetic acid ( edta ) [ 34 , 5759 , 61 , 63 , 6569 , 71 , 74 , 7678 , 81 , 82 ] . reviewing literature on use of several microorganisms in pdt studies , authors could not evaluate in vivo studies in those terms , because no attempt was made to identify bacterial flora during culture process [ 35 , 58 , 73 , 75 ] in four of six studies . only garcez et al . , 2010 , and juri et al . , 2014 , established microbiological identification . among all studies , we analysed 23 studies ( all in vitro and ex vivo ) , and from those , 20 ( 87% ) elected enterococcus faecalis as substract to quantify antimicrobial pdt effectiveness . e. faecalis is a gram - positive facultative anaerobe commonly detected in asymptomatic , persistent endodontic infections . this finding can be explained by various survival and virulence factors expressed by e. faecalis , including its ability to compete with other microorganisms , invade dentinal tubules , and resist nutritional deprivation . e. faecalis was used not only in planktonic suspensions , but also in form of biofilms and the most common strain selected was atcc29212 . however , biofilm maturation time did not follow a linear pattern ; besides , a huge discrepancy exists . some authors used young biofilms with range of 2 [ 60 , 68 ] , 4 , and 7 days [ 81 , 82 ] very distinct from mature biofilms performed with biofilms of 21 [ 61 , 66 ] , 28 [ 5 , 69 , 72 , 77 ] , and 70 days . according to kishen and haapasalo 2010 , a mature biofilm is considered when maturation period is equal to or higher than 21 days and only 7 ( 30% ) studies [ 5 , 59 , 61 , 66 , 69 , 72 , 77 ] respected this mature biofilm criteria . apart from e. faecalis of note , in literature , the first pdt in vitro study was performed by seal et al . 2002 in root canals infected with streptococcus intermedius ( gram - positive facultative anaerobe ) biofilm with 2 days of maturation using tbo as ps and a helium - neon laser as light source . pdt , a technique with potentially significant antimicrobial properties , is a fairly recent approach in endodontic disinfection protocols . while the oral applications of pdt have been extensively tested , variations in study type and design limits the ability to synthesize or pool the available quantitative data , thereby permitting a formal meta - analysis and a systematic review . furthermore , many of the studies quantitatively measuring the degree of bacterial kill fail to report baseline bacterial counts or concentrations , thus limiting the ability to assess the bactericidal efficacy of pdt . considering this apparent variation in reporting results among the studies analysed , it is difficult to provide a definitive assessment of the research question posed in this review . it is important to mention that pdt efficacy is shown in cfu or in percentage and logarithm ( in form of log10 ) ; nonetheless , authors state this is pointless without the perception of the initial concentration . as an example , if we have an initial sample from a root canal of 10 microorganisms and if after pdt approach we had 10 , statistically , 99% were killed , but there are still 100000 microorganisms left inside the root canal . considering the variation in units at outcomes , the final results analysis is difficult . even though pdt has significant advantages ( cited in section 1 ) , potential adverse events as tooth discoloration have been reported previously in root canal treatment when mb and tbo were used as ps . it is also important that future clinical studies clearly report adverse events associated with pdt so that an estimation of the benefit - to - risk ratio from the use of pdt is feasible . nonetheless , there were no adverse effects mentioned in the included studies of the current review . pdt outcomes in literature have been reported by the dual combination of ps and a visible light source in the presence of oxygen ; however , recently , lins de sousa et al . analysed that twice - daily blue light of 420 nm , energy density of 72 jcm , and irradiation time of 776 s without ps are a promising approach in the inhibition of five days ' streptococcus mutans matrix - rich biofilm development . it has remarkably inhibited the production of insoluble eps , which is responsible for the scaffold of the extracellular biofilm matrix . the authors suggest that this evidence is very important to improve standardization in pdt procedures in the total absence of light as the evaluation of ps dark toxicity in some studies reviewed did not address this important issue . in the literature , residual systemic photosensitization has also been reported as a potentially adverse event associated with the use of intravenous ps ; but this effect appears to not be associated with oral applications of pdt . the role of pdt in root canal disinfection has been tested using several combinations of ps and light sources and has shown divergent results and these studies have revealed several limitations associated with antimicrobial pdt . for successful pdt to affect significant reduction or eradication of microorganisms , a ps is required which will show enough affinity for microorganisms without catalyzing photodamage to host tissues , a light source at a wavelength that can penetrate tissues ( 630700 nm ) , and sufficient oxygenation to produce a level of reactive oxygen species ( ros ) necessary to induce photodynamic lipid peroxidation and , as a consequence , necrosis and cell death . if there is photodamage to both tissues and microorganisms , efficacy will be suboptimal . microorganisms in the root canal flora and their growth mode were found to influence their susceptibility to pdt in a dose - dependent manner and biofilms can be difficult to eradicate not only because of their effect as barriers to ps uptake , but also their ability to diffuse or attenuate light in the root canal dentinal tubules . even dentin , dentin matrix , pulp tissue , bacterial lipopolysaccharides , and bovine serum albumin were found to significantly decrease pdt antimicrobial efficacy and , as a consequence , an effort to enhance the pdt by nanoparticle - based technology appears promising . other strategies include the use of a ps solvent , efflux pump inhibitors , or photoactivated functionalized chitosan nanoparticles for disinfection and stabilization of the dentin matrix . because the application of pdt for additional reduction of the microbial load of root canal systems seems promising , it would be beneficial to identify the ideal combination of ps and light wavelength in preclinical studies and conduct future randomized controlled trials to test the effect of pdt on root canal disinfection in various indications . pdt has been used thus far without a consensus - based , well - defined protocol , and therefore still remains at an experimental stage waiting for further optimization . limited clinical information is currently available on the use of pdt in root canal disinfection . currently , the level of evidence of available clinical studies to answer this question is low . nevertheless , the results of this review suggest , based primarily on available in vivo studies , that pdt could perform well as an antimicrobial adjuvant . pdt appears to be a promising antimicrobial platform so further studies are warranted to optimize protocols using standardized laser and ps parameters to assess the pdt efficacy . therefore , within the limits of the present review , one may conclude that the efficacy of pdt remains questionable , but promising . it is further suggested that an additional potential benefit from the use of pdt in root canal disinfection may exist where highly resistant bacteria are present in the root canal space , thus affecting the treatment prognosis . further research is necessary to establish the appropriate pdt parameters allowing adequate antimicrobial action without harmful host side effects .

it has been reported that thiopurines are effective at maintaining remission in steroid - dependent or steroid - refractory cd and uc and have steroid - sparing effect.123 they have also shown efficacy in preventing postoperative recurrence of cd.45 although their clinical implications are limited , thiopurines remain a mainstay of treatment of patients with ibd in the biologic era . however , possible toxicities such as bone marrow suppression , hepatotoxicity , and increased risk of opportunistic infections and malignancies interfere with the use of these potent drugs in clinical practice . at present , there exist several questions regarding the optimization of thiopurine therapy . to balance the efficacy with safety and tolerability , thiopurine s - methyltransferase ( tpmt ) pretreatment screening , prudent dose escalation , and careful monitoring using novel biomarkers have been suggested.678 however , most of the current guidelines for thiopurine use in ibd have been established based on western data . it has been widely accepted that notable differences in the context of epidemiology , genetics , and clinical characteristics exist between western countries and east asia , including korea.910 therefore , there is a definite need to establish management strategies that are more suitable for korean patients . for this reason , the korean association for the study of intestinal diseases ( kasid ) developed a set of consensus statements on thiopurine use in ibd . the aim of these statements is to offer guidance to clinicians on the appropriate use of thiopurines in the management of ibd . the committee for the development of consensus statements comprised five members of kasid ( working group ) . the committee conducted extensive literature review regarding thiopurine use in ibd and developed initial statements . the statements consisted of four parts : ( 1 ) pre - treatment evaluation and management strategy , ( 2 ) treatment with thiopurines , ( 3 ) safety of thiopurines , and ( 4 ) monitoring of side effects or efficacy of therapy . the quality of evidence and classification of recommendation was assessed according to the grading of recommendation assessment , development and evaluation ( grade ) system.11 in this system , the quality of evidence of each statement was categorized as high , moderate , low , or very low . evidence based on randomized controlled trials was initially classified as high - quality evidence , but the rating could be downgraded for several reasons including study limitations , inconsistency of results , indirectness of evidence , imprecision , and reporting bias . although data from observational studies ( e.g. , cohort and case - control studies ) were initially classified as low - quality evidence , the rating could be upgraded if the magnitude of the treatment effect is very large , if there is evidence of a dose - response relation , or if all plausible biases were found to decrease the magnitude of an apparent treatment effect.1112 based on the grade system , the strength of recommendations was classified as strong or weak , determined by four key factors including the balance between the desirable and undesirable effects , quality of evidence , values and preferences , and costs ( resource allocation).13 the definitions of the levels of evidence and recommendations are summarized in table 1 . prior to the consensus meeting , consensus statements were circulated to academic gastroenterologists with experience in managing patients with ibd and were revised in accordance with their comments and opinions regarding each statement . the consensus meeting was held on april 18 , 2015 in seoul , korea , wherein 38 ibd experts participated . during the meeting , the working group presented statements and all participants voted on their level of agreement regarding each statement . a consensus statement was accepted if at least 75% of participants voted 1 ( strongly agree ) or 2 ( agree ) on a scale of 1 to 5 ( with 3 , 4 , and 5 indicating uncertain , disagree , and strongly disagree , respectively ) . if a statement was not accepted , the wording of the statement was discussed and revised , and then re - voting was conducted . thiopurine - induced leukopenia is more common in east asians , namely , chinese , japanese , and koreans , compared to caucasians . quality of evidence : moderate classification of recommendation : strong level of agreement : strongly agree 55% , agree 40% , uncertain 5% thiopurines have been widely used for the treatment of patients with ibd . recommended dosages for the treatment of ibd are 2.0 - 2.5 mg / kg / day for aza and 1.0 - 1.5 mg / kg / day for 6-mp.67 however , one of the major drawbacks of the use of thiopurines is the development of leukopenia ( defined as white blood cell [ wbc ] count < 3,000/mm ) , occurring in up to 5% of caucasian patients with ibd treated with these agents.141516 interestingly , the occurrence of thiopurine - induced leukopenia is considerably high in asians.171819202122 in a study by lee et al.,17 thiopurine - induced leukopenia was observed in 116 ( 31.2% ) of 372 korean patients with cd at a median aza dose of 1.34 mg / kg / day . in a study by yang et al.,18 leukopenia was noted in 346 ( 35.4% ) of 978 korean patients with cd at a median aza dose of 1.70 mg / kg / day . in another korean multicenter study,19 leukopenia developed in 110 ( 39.6% ) of 278 patients with ibd at a mean aza dose of 1.80 mg / kg / day . in two japanese ibd cohorts with wild - type tpmt,2021 leukopenia was observed in 18 ( 15.8% ) of 114 patients and 7 ( 10.0% ) of 70 patients , although the total daily dose of aza was only 50 mg in the majority of patients . in a study by fangbin et al.,22 leukopenia ( wbc < 3,500/mm ) was noted in 36 ( 18.1% ) of 199 chinese patients with ibd . since all these east - asian studies used doses lower than the recommended dosage of aza , more frequent and severe leukopenia is expected with standard doses of thiopurines in this population . assessment of the tpmt genotype or enzyme activity before initiating thiopurine therapy is of limited value in east - asian populations compared to that in caucasians . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 29% , agree 55% , uncertain 16% aza and 6-mp are metabolized by tpmt . tpmt mutations that result in decreased enzymatic activity are associated with a greater risk of thiopurine - induced leukopenia.23242526 therefore , in order to mitigate the risk of leukopenia with thiopurines , the us food and drug administration recommends an assessment of tpmt status prior to initiating therapy.7 however , only about a quarter of ibd patients with thiopurine - induced leukopenia carry a tpmt mutation , thus raising questions on the utility of tpmt pre - testing.272829 moreover , while the frequency of tpmt mutations is lower in asians ( 1 - 3%)1819202122 than that in caucasians ( ~10%),30 the occurrence of thiopurine - induced leukopenia in asians is considerably high , as described in the statement 1.171819202122 consequently , tpmt mutations are present in only 0 to 5.6% of asian patients with ibd who develop thiopurine - induced leukopenia.1819202122 in addition , homozygotes for tpmt mutant alleles are extremely rare ( ~0.01% ) in the japanese population 20 whereas their frequency is about 0.6% among caucasians.31 taken together , assessment of tpmt genotype / phenotype prior to thiopurine therapy is of limited value in east - asian populations , when compared with caucasians . gradual increase in the dosage of thiopurines over several months until the target dose is usually preferable to beginning the medication at the target dose at the onset , in east asians . quality of evidence : low classification of recommendation : weak level of agreement : strongly agree 58% , agree 42% the recommend target dose of aza and 6-mp for ibd treatment is 2.0 - 2.5 mg / kg / day and 1 - 1.5 mg / kg / day , respectively , if the patients can tolerate the drugs.67 however , the best approach to attain the target dose has not been established yet . the traditional " dose escalating approach " is to initiate the therapy at 50 mg daily , then increase the dose by 25 mg every 1 - 2 weeks to a target dose along with monitoring for leukopenia and other potential adverse events.7 the main concern regarding this approach is under - dosing of patients with a suboptimal response rate . an alternate approach is to start immediately at the full standard dose based on tpmt activity . according to this approach , patients with normal tpmt activity receive the standard dose while the patients with intermediate tpmt activity receive 50% of the standard dose . however , patients with low tpmt activity ( 0.3% of general population ) should not be treated with thiopurines.32 interestingly , a questionnaire - based survey showed that 33% of us gastroenterologists initiated therapy at 50 mg of aza , while 28% administered a full dose of aza at 2.5 mg / kg / day , in clinical practice.33 interestingly , the usual approach to prescribing thiopurines in east asia is to start at a low dose and to gradually increase the dose.34353637 according to a survey on clinical practice patterns in the treatment of ibd in korea , 80% of the responders initiated aza at 50 mg / day , 68% increased the dose by 25 mg , and 56% increased the dose every 4 weeks.34 this gradual dose increment policy could be useful for reducing aza - induced myelotoxicity.35 however , it may delay the time to clinical response . in china , a dose step - up strategy is typically carried out by gradually increasing the dose of aza to the target dose under close monitoring of laboratory results and clinical response.36 in japan , lower doses of aza ( 50 mg / day or 0.6 - 1.2 mg / kg / day ) are employed for the treatment of patients with ibd.2037 unlike western data , several studies have suggested that lower doses of aza or 6-mp may be effective and safe for the treatment of patients with ibd in east asia.17363738 however , these studies did not compare the efficacies of lower doses with those of standard doses ; moreover , they did not analyze the 6-thioguanine nucleotide ( 6-tgn ) levels . nevertheless , considering higher incidences of myelotoxicity 171819202122 and limited value of the assessment of tpmt genotypes,1920212227282939 the dose escalating approach starting with low doses can be an effective and safe strategy in east asians . determination of nudt15 genotypes before initiating thiopurine therapy may identify patients with a predisposition to the development of thiopurine - induced early leukopenia . quality of evidence : moderate classification of recommendation : strong level of agreement : strongly agree 13% , agree 76% , uncertain 11% as described in statement 2 , assessment of the tpmt genotype or enzyme activity may play a limited role in the prevention of thiopurine - induced leukopenia in east asians . recently , a non - synonymous single nucleotide polymorphism in nudt15 variant was identified as a significant risk factor for thiopurine - induced early leukopenia in korean patients with cd.18 the nudt15 allele encoding p.arg139cys was found in 89.4% of the early leukopenia cases but was found in only 6.8% of the controls , suggesting that the presence of the nudt15 allele had a sensitivity of 89.4% and specificity of 93.2% to early leucopenia . although rare , this single nucleotide polymorphism was also associated with thiopurine - induced leukopenia in caucasians.18 the frequency of nudt15 risk allele is much higher in east asians than that in caucasians ( 10.4% in koreans , 7% in japanese , 13% in chinese , and 2% in an admixed american population).18 the results of this study were reproduced by a study in children with acute lymphoblastic leukemia conducted in the united states.39 therefore , pre - treatment determination of nudt15 genotypes may help to identify patients susceptible to thiopurine - induced early leukopenia in diverse populations , especially in east asians . there is not enough evidence to recommend thiopurine monotherapy for induction of remission in active cd or uc . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 42% , agree 53% , uncertain 5% the data for thiopurines for remission induction in active uc is limited.4041 in a meta - analysis of 4 randomized controlled trials ( n=89 ) evaluating the efficacy of aza/6-mp for the induction of clinical remission of uc , the mean efficacy of aza was not superior to placebo or 5-aminosalicylate ( 75% vs. 64% , respectively ; or , 1.59 ; 95% ci , 0.59 - 4.29 ; p=0.13).41 thiopurines may be effective for inducing remission in active cd as compared to placebo ( or , 2.43 ; 95% ci , 1.62 - 3.64).42 however , a recent cochrane meta - analysis including 11 randomized trials reported that thiopurines offered no significant advantage over placebo for induction of clinical remission in active cd ( 48% vs. 37% , respectively ; rr , 1.23 ; 95% ci , 0.97 - 1.55).43 usually , thiopurines have been limited in the use of remission induction in active cd or uc because of the delay in the onset of their action.44 thiopurines may take 3 - 6 months to achieve full clinical efficacy.45 therefore , in patients with active cd or uc requiring rapid symptom relief , concomitant therapy with systemic corticosteroids and thiopurines is a more reasonable approach . however , the addition of thiopurines to corticosteroids makes no benefit in terms of remission induction.44 thiopurines have the benefit in corticosteroid sparing effect for active cd with a pooled or of 3.69 ( 95% ci , 2.12 - 6.42).42 thiopurines are effective for the maintenance of remission in patients with cd and uc . quality of evidence : high classification of recommendation : strong level of agreement : strongly agree 66% , agree 32% , uncertain 2% thiopurines are effective for maintenance of remission in both uc and cd.23 aza appears to be more effective than placebo for the maintenance of remission in uc.3 according to a meta - analysis of 4 studies including 232 patients with uc , 44% ( 51/115 ) of patients administered aza failed to maintain remission compared to 65% ( 76/117 ) of placebo - administered patients ( rr , 0.68 ; 95% ci , 0.54 - 0.86).3 aza or 6-mp may be effective as maintenance therapy in patients with uc who have failed to respond or can not tolerate 5- aminosalicylate and in patients who require repeated courses of corticosteroids . in case of patients with cd , aza or 6-mp also had a positive effect in maintaining remission.124647 a meta - analysis of 8 studies including 503 patients with cd revealed that the overall remission rate was 71% ( 147/208 ; 95% ci , 64 - 77% ) for aza treatment compared to that for placebo ( 55% [ 141/255 ] ; 95% ci , 49 - 61%).2 aza ( 1.0 - 2.5 mg / kg / day ) is effective in reducing the risk of disease recurrence over a 6-month to 2-year period . higher doses of aza ( 2.5 mg / kg / day ) are more effective than the lower doses ( 1.0 or 2.0 mg / kg / day ) in preventing disease recurrence . in addition , aza has corticosteroid - sparing effects that reduce corticosteroid - related side effects . previously , a korean study also reported that complete withdrawal of corticosteroids was achieved in 70.9% of patients with cd receiving treatment with aza.17 aza or 6-mp may also benefit patients dependent on corticosteroids or in cases where corticosteroid treatment has failed . among korean patients with steroid - dependent uc , the 3-year success rate was higher in the aza therapy group ( 71.2% ) than that in the aza intolerance group ( 25.0%).38 however , withdrawals due to adverse events were more commonly observed in patients treated with aza ( peto or , 3.74 ; 95% ci , 1.48 - 9.45 ) compared to placebo.2 optimal duration of treatment with thiopurines is uncertain . prolonged or indefinite use of thiopurines may be considered to maintain remission . the risk and benefits of continuing thiopurine therapy should be balanced and discussed with individual patients . quality of evidence : low classification of recommendation : weak level of agreement : strongly agree 68% , agree 29% , uncertain 3% conclusive evidence suggests that thiopurines are effective for maintenance of remission in both cd and uc.23 well - tolerated and effective therapy is generally continued to maintain the remission of ibd . there is limited data regarding the factors predicting responses to aza and the uncertainty regarding the optimal duration of treatment.48 in patients with cd , several studies showed relapse rates of 14 - 41% at 1 year after cessation of thiopurines with a cumulative increase with time.495051 furthermore , patients with cd who discontinued aza after more than 3 years of efficacious treatment had a higher probability of relapse than those who continued therapy.52 a recent meta - analysis showed that there is a clear benefit of continuing aza/6-mp therapy for at least 18 months to maintain remission in patients with cd.53 in patients with uc , relapse rates after cessation of aza treatment were reported to be 35 - 77% and 65 - 75% at 1 and 5 years , respectively.165455 there was no difference in the relapse rates between cd and uc . the duration of aza treatment did not affect the relapse rates after cessation of treatment ( p=0.68).16 according to the european crohn 's and colitis organisation ( ecco ) guidelines,56 for patients with cd who have been treated with thiopurines as part of maintenance therapy , discontinuation may be considered after 4 years of remission . to summarize these data , thiopurines should be used for at least 18 months after achieving remission and could be continued for over 4 years . therefore , considering the high cumulative relapse rates after cessation of thiopurine treatment , risk stratification is an important issue for patients with ibd receiving thiopurines . meanwhile overall , around 10 - 28% of patients reported side effects and of which 50 - 80% discontinued the thiopurines as a result.57 in addition to risk factors for disease relapse , adverse events with long - term use such as serious infections and malignancies must be taken into account . thus , benefits and risks of continuing thiopurines should be considered and discussed with each patient individually . the combination of anti - tumor necrosis factor ( tnf)- agents and thiopurines is more effective than that of monotherapy with anti - tnf- agents or thiopurines alone in inducing remission in patients with moderate to severe cd . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 26% , agree 58% , uncertain 16% quality of evidence : high classification of recommendation : strong level of agreement : strongly agree 70% , agree 24% , uncertain 6% several studies have reported the effectiveness of combination of anti - tnf agents with thiopurines in case of rheumatic diseases and ibd . thiopurines improved the pharmacokinetics of anti - tnf agents and decreased the formation of anti - drug antibodies.58 in the sonic ( the study of biologic and immunomodulator naive patients in crohn 's disease ) trial that included immunosuppressant - naive cd patients , the corticosteroid - free remission rates and mucosal healing rates were higher in those treated with a combination of infliximab plus aza than those treated with infliximab or aza alone.59 the corticosteroid - free remission rates at week 26 were 57% in those receiving combination therapy , 44% in those receiving infliximab alone , and 30% in patients receiving aza alone . at week 50 , the corticosteroid - free remission rates were 72% , 61% , and 55% . additionally , mucosal healing rates were evaluated at week 26 and they were 44% , 30% , and 16.5% , respectively.59 in the getaid ( groupe d'etude therap - utique des affections inflammatoires du tube digestif ) study , infliximab plus aza/6-mp was found to be more effective than aza/6-mp alone for the induction of remission in patients with corticosteroid - dependent cd.60 in case of adalimumab , a recent meta - analysis study demonstrated that adalimumab monotherapy was inferior when compared to combination therapy with adalimumab plus thiopurines ( or , 0.78 ; p=0.02 ) for induction of remission in patients with cd . however , the rate of remission at 1 year and the need for dose escalation was similar in both groups.61 therefore in patients with moderate to severe cd , especially in case of immunosuppressant - naive patients with cd , a combination therapy with anti - tnf and thiopurines seems to be preferred mode of treatment in order to allow the patients to achieve the highest rate of rapid disease control and limited tissue damage . however , the benefit of combination therapy in immunosuppressant treatment - failed patients remains unclear . the combination of anti - tnf- agents and thiopurines is more effective than that of monotherapy with anti - tnf- agents or thiopurines alone in inducing remission in patients with moderate to severe uc . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 18% , agree 66% , uncertain 16% quality of evidence : high classification of recommendation : strong level of agreement : strongly agree 55% , agree 42% , disagree 3% the main question that remains to be answered in this clinical situation is whether combination therapy with infliximab and thiopurines is clearly superior to monotherapy with infliximab or thiopurines alone , in patients with uc . recently , the uc success ( efficacy and safety of infliximab and azathioprine monotherapy or in combination in moderate to severe uc ) trial was performed to provide a suitable explanation for the above question.62 this study was a randomized , double - blind clinical trial for evaluating the efficacy and safety of 16 weeks of infliximab plus aza , infliximab or aza monotherapy in patients with moderate to severe uc.62 at week 16 , a greater proportion of patients in the infliximab plus aza achieved corticosteroid - free remission ( 39.7% ) compared to 22.1% receiving infliximab alone ( p=0.017 ) and 23.7% receiving aza alone ( p=0.032 ) . mucosal healing at week 16 occurred in 62.8% of patients receiving infliximab plus aza , compared to 54.6% receiving infliximab alone ( p=0.295 ) and 36.8% receiving aza alone ( p=0.001 ) . adverse events that required discontinuation of therapy were higher in the aza alone ( 8% ) than in the infliximab alone ( 3% ) or the infliximab plus aza group ( 4% ) . although there are no high quality reports regarding the superiority of combination therapy except for the uc success trial , infliximab - based combination therapy can be more therapeutically useful in patients with moderate to severe uc who previously do not appear to respond adequately to corticosteroid therapy . the combination of adalimumab and immunomodulator appeared mildly superior to adalimumab monotherapy for the induction of remission in cd.61 however , it is still unclear in case of uc . selection between anti - tnf- monotherapy and combination therapy with an immunomodulator may be individualized based on the risk of relapse and adverse events . quality of evidence : low classification of recommendation : no level of agreement : strongly agree 32% , agree 65% , uncertain 3% as described in statements 8 and 9 , combination therapy with infliximab and thiopurines is more effective than monotherapy with either agent alone in inducing corticosteroid - free clinical remission and mucosal healing in patients with cd and uc who are naive to either agent.5962 meanwhile , the clinical benefits of continuing immunomodulators in patients with ibd who are refractory to these drugs when starting anti - tnf therapy are debatable . however , combination therapy with anti - tnf agents and immunomodulators may increase the risk of serious infections and malignancies . in a study with longitudinal cohort of 8,581 cd patients,63 monotherapy with steroids , immunomodulators , or anti - tnf agents was associated with an increased risk of tuberculosis , candidiasis , herpes zoster , and sepsis as compared to patients not receiving these medications . in contrast , according to the treat ( crohn 's therapy , resource , evaluation and assessment tool ) registry,64 cd severity and use of prednisone or narcotic analgesics showed higher risks of serious infection , although an increased risk was also observed with infliximab . moreover , treatment with immunomodulators including thiopurines and methotrexate was not a significant predictor of serious infection , and combination therapy with infliximab and immunomodulators did not increase the risk of infection , compared with infliximab monotherapy . therefore , relationship between combination therapy with infliximab and immunomodulators and development of serious infections remains unclear . the risk of neoplasm in ibd patients receiving anti - tnf therapy is controversial.6566 the combination of anti - tnf agents and immunomodulators is associated with an increased risk of malignancies such as lymphoma and non - melanoma skin cancer.676869 the risks of these cancers are largely driven by the use of concomitant immunomodulators , particularly thiopurines.70 in order to decide between the combination therapy versus monotherapy approach , we should weigh the increased risk of complications induced by combination therapy against the increased risk of relapse observed with monotherapy . according to a decision analytic model , the benefits of combination therapy with infliximab and aza would outweigh the risks in cd patients who are naive to either agent , unless serious infections occurred in 20% or more of the population or lymphoma in 3.9% or more.71 old age ( > 65 years ) is associated with a greater risk of serious infections and lymphoproliferative disorder.7273 hepatosplenic t - cell lymphoma usually occurs in young men ( < 35 years ) receiving thiopurine therapy for more than 2 years.74 factors associated with the risk of relapse after cessation of concomitant immunomodulators include increased inflammatory markers , evidence of mucosal activity on endoscopy , short duration in remission before stopping therapy , and undetectable trough levels of anti - tnf.70 therefore , withdrawal of concomitant immunomodulators may be considered in patients in deep remission for a considerable period and with detectable anti - tnf trough levels , especially in elderly patients or young male patients . currently , there is no consensus on the duration of combination therapy with an anti - tnf agent and an immunomodulator . use of thiopurines is associated with an increased risk of lymphoma and non - melanoma skin cancers . quality of evidence : moderate classification of recommendation : strong level of agreement : strongly agree 51% , agree 49% according to a recent meta - analysis by kotlyar et al.,75 the overall standardized incidence ratio ( sir ) for lymphoma was 4.92 ( 95% ci , 3.10 - 7.78 ) in patients with ibd exposed to thiopurines : 2.80 ( 95% ci , 1.82 - 4.32 ) in 8 population studies and 9.24 ( 95% ci , 4.69 - 18.2 ) in 10 referral studies . in 8 population studies , an increased risk was noted among current users ( sir , 5.71 ; 95% ci , 3.72 - 10.1 ) but not among former users ( sir , 1.42 ; 95% ci , 0.86 - 2.34).75 young male patients ( < 30 years ) had the highest sir , and aged patients ( > 50 years ) showed the highest absolute risk.75 according to a recent meta - analysis involving 60,351 ibd patients , the pooled adjusted hazards ratio of developing non - melanoma skin cancers after exposure to thiopurines was 2.28 ( 95 % ci , 1.50 - 3.45).76 however , the results of this meta - analysis must be interpreted carefully owing to a marked heterogeneity between studies . there is not enough evidence to suggest that the modestly increased risk of lymphoma and non - melanoma skin cancers outweighs the benefit of thiopurines in ibd.6776 use of thiopurines is not associated with an increased risk of postoperative complications . quality of evidence : low classification of recommendation : weak level of agreement : strongly agree 30% , agree 67% , uncertain 3% postoperative complications in ibd include abdominal sepsis , wound problem , anastomotic leakage , early reoperation as well as complicated systemic diseases leading to increased morbidity and mortality . in case of cd , a study by colombel et al.77 reported that early postoperative complications did not increase in patients with cd treated perioperatively with immunosuppressive agents . however , in a study by myrelid et al.,78 preoperative thiopurine therapy was associated with postoperative intra - abdominal septic complications in case of abdominal surgery for patients with cd . as for uc , in a study by schaufler et al.,79 preoperative exposure to thiopurines was not associated with increased postoperative complications in a cohort undergoing colectomy for uc or ibd - u . recently , a meta - analysis involving 21 studies with 6,899 ibd patients evaluated whether the preoperative use of immunosuppressive agents was associated with increased postoperative complications in patients with ibd.80 of these 21 studies , 8 studies with 1,674 patients reported the impact of preoperative thiopurines on postoperative outcomes . the pooled risk ratio estimates for total complications and infectious postoperative complications were 0.97 ( 95% ci , 0.69 - 1.36 ) and 1.23 ( 95% ci , 0.66 - 2.29 ) , thus suggesting no association between preoperative use of thiopurines and postoperative complications.80 however , most studies were retrospectively designed and there were large variations in the patient populations and outcome definitions.80 thiopurines are considered to be safe and well tolerated during pregnancy . discontinuing thiopurine therapy during pregnancy may precipitate a flare resulting in adverse neonatal outcomes . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 16% , agree 71% , uncertain 13% the peak incidence of ibd occurs in women of childbearing age . management of patients with ibd during pregnancy requires a challenging balance between optimal disease control and drug safety considerations . fears about the potential harm of ibd medications to the developing fetus are very common . in a survey conducted in australia , 36.1% of participants believed that any form of ibd medication was harmful to the unborn fetus.81 however , discontinuation of therapy may result in disease relapse during pregnancy , which carries a greater risk of adverse fetal outcomes ( a higher rate of fetal loss , preterm birth , and low birth weight ) , according to a number of reports.828384858687 conflicting data exist regarding the association between thiopurine use for ibd treatment in pregnancy and adverse pregnancy outcomes . a few studies reported an increased risk of fetal loss , preterm delivery , low birth weight , and congenital abnormalities when thiopurines were used during pregnancy.888990 however , these outcomes might have been caused by the underlying disease rather than thiopurines . a majority of recently conducted controlled or cohort studies have not shown an increase in congenital abnormalities.9192939495 according to a recent pharmacological study conducted on thiopurines in pregnant patients with ibd , although unborn children were exposed in utero to the pharmacologically active thiopurine metabolites 6-tgn , no major teratogenicity was observed.96 in another study , thiopurine use during pregnancy did not affect long - term development or immune function of children up to 6 years of age.97 moreover , two recent meta - analyses did not demonstrate an increased risk of fetal loss , low birth weight , or congenital abnormalities despite intrauterine exposure to thiopurines.9899 however , an association with preterm birth was noted in one out of the two meta - analyses studies.98 in addition , 60% of newborns exposed to thiopurines in utero were anemic at birth.96 existing guidelines suggest that thiopurines should not be discontinued during pregnancy.100101102103 in a worldwide survey among 175 ibd experts , 155 ( 89% ) physicians replied that they would continue aza therapy throughout pregnancy.4 the potential risks and benefits of thiopurine therapy should be discussed with a patient , ideally prior to conception . breastfeeding could be advised for women on thiopurine maintenance therapy willing to nurse their infants . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 23% , agree 64% , uncertain 13% treatment with thiopurines is widely used to maintain remission in ibd . breastfeeding by patients with ibd patients on thiopurines is probably safe because the presence of only trace amounts of aza/6-mp metabolites has been noted in breast milk . in a prospective study , the concentration of 6-mp was measured in 31 breast milk samples collected from 10 mothers receiving aza.104 6-mp was detected in low concentrations ( 1.2 and 7.6 ng / ml , as compared to therapeutic immunomodulator level of 50 ng / ml in serum ) in two samples , but was not detected in any of the other 29 samples.104 additionally , aza/6-mp metabolites were undetectable in the neonatal blood.97104 the majority of thiopurine metabolites are excreted in milk in the first 4 hours after intake of the drug.105 hence , mothers should be counseled to use a breast pump 4 hours after medication intake to discard the first portion of milk produced after aza intake in order to minimize the infant 's exposure to the drug . breastfeeding by ibd patients undergoing thiopurine therapy did not affect long - term development or immune function . one retrospective study showed no differences in infection rate in children breastfed by mothers receiving thiopurines for ibd as compared to children breastfed by mothers without immunomodulator therapy.92 another recent observational study showed no differences in any of the global medical and psychosocial health status in 9 breastfed infants ( for median 7 months , range 3 - 13 months ) and the formula - fed group.106 thiopurines can be administered safely to women with ibd during lactation . therefore , breastfeeding should be recommended after considering its beneficial therapeutic effects for the nursing mother with ibd . appropriate counseling for lactation should be available for all lactating women with ibd in order to discuss the risks and benefits of thiopurine therapy . close follow - up of full blood counts is recommended in all patients taking thiopurines . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 28% , agree 67% , uncertain 5% thiopurines have been reported to increase the risk of myelotoxicity , particularly leukopenia . myelotoxicity is one of the more common dose - dependent adverse effects with potentially serious clinical consequences . the use of complete blood counts ( cbc ) to monitor bone marrow suppression is of particular importance.107 most cases of severe leukopenia occur abruptly early on in treatment.107 in a study by lewis et al . , the incidence of severe leukopenia ( wbc < 1,000/mm ) was highest in the first 8 weeks of thiopurine therapy with the median time from onset of therapy to first documentation of severe leukopenia being 24.5 days.108 there is currently no clear consensus on optimal frequency of blood monitoring.109 several monitoring guidelines have been proposed.15107110111 a western study stated that a cbc should be obtained weekly for one month , biweekly for the second month , monthly for four months and , bimonthly when the patient is stable after six months of treatment . although further studies are needed in order to better define the optimal interval of cbc monitoring , it may be scheduled biweekly for the first two months , and then every 4 - 12 weeks thereafter , in general . the dose adjustment of thiopurines through monitoring of 6-tgn and 6-methylmercaptopurine ( 6-mmp ) levels is expected to improve efficacy and reduce side effects in patients treated with thiopurines . quality of evidence : moderate classification of recommendation : weak level of agreement : strongly agree 18% , agree 69% , uncertain 13% the variation between clinical efficacy and side - effect profile of thiopurines is may be attributed to individual differences in drug metabolism . 6-mp and its prodrug aza are converted to 6-tgn , which is the main active metabolite responsible for therapeutic efficacy and myelotoxicity . on the other hand , metabolism of these products also yields 6-mmp , high levels of which are associated with increased hepatotoxicity . these findings have led to strategies for advantageously shifting thiopurine metabolism toward optimal 6-tgn levels while decreasing 6-mmp levels in an attempt to benefit a greater portion of patients undergoing thiopurine treatment . although the relationship between 6-tgn levels and efficacy was first described in patients with ibd in 1996,109 this correlation was confirmed in the landmark study in 2000 by dubinsky et al.,112113 which demonstrated a positive correlation of therapeutic response with 6-tgn levels in 92 pediatric patients with ibd . the frequency of therapeutic response increased at 6-tgn levels > 235 pmol/810 red blood cells ( rbc ) while hepatotoxicity correlated with elevated 6-mmp levels ( > 5700 pmol/810 rbc).113 since then , several prospective studies have reported a correlation between 6-tgn levels and clinical response.114115116117 a meta - analysis of 12 studies with 941 patients concluded that patients with 6-tgn levels above 230 - 260 pmol/810 rbc were more likely to be in remission than those below the threshold value.8 a more recent pooled analysis including 17 studies of 2,049 patients showed that the pooled or for clinical remission among patients with 6-tgn levels over a cut - off value between 230 and 260 pmol/810 rbc was 3.15.118 although the dose adjustment of thiopurines through monitoring of 6-tgn and 6-mmp levels is expected to improve efficacy and reduce side effects in patients treated with thiopurines , further studies are needed in order to confirm these findings . for decades , thiopurine agents have been a mainstay in treating ibd and will play an important role in the future . however , complex metabolism and various side effects limit their successful application in clinical practice . thiopurine - induced leukopenia is especially very common and prescreening for tpmt has a limited value in korean patients with ibd . in addition , use of novel biomarkers such as nudt15 , may help identify patients who are at a high risk for thiopurine - induced leukopenia . at present , optimal duration of thiopurine treatment in addition to long - term outcomes of combination therapy with anti - tnf agents although thiopurine treatment during pregnancy and lactation is considered safe and well tolerated , it is important to balance the risks and benefits .

as the incidence of obesity increases , its impact on the care and outcomes of the critically ill patient has become an issue . there are numerous concerns when caring for obese patients , which include the impact on survival to the ability of the care provider to move the patient . in terms of survival of these patients , both positive and negative outcomes have been associated with obesity , specifically body mass index , which has been termed the ' obesity pdox ' . this inconsistency in survival has stimulated an increased interest in the role of adipose tissues in critical illness . adipose tissue has generally been viewed as an inert tissue that was a store for excess energy . in the past two decades , adipose tissue has become recognized as a dynamic tissue involved in the regulation of metabolism , the inflammatory response , coagulation and insulin sensitivity , all of which are major elements in the response to critical illness . langouche and colleagues [ 1 - 3 ] have demonstrated that adipocytes undergo morphological changes in patients with critical illness , resulting in smaller adipocytes with increased glucose and lipid storage capabilities and increased accumulation of m2 phenotype macrophages . these changes are suggested to impart beneficial effects improving patient outcomes . in their present study , marques and colleagues postulated that these changes were independent of the nutritional status of the subject . they employed the long - term cecal ligation and puncture ( clp ) mouse model of sepsis and made two comparisons . the first is the comparison of healthy animals to animals with clp who were fed approximately 66% of their daily requirement . the clp animals demonstrated a significant reduction in total body fat , smaller adipocytes and increased deposition of m2 macrophages , changes similar to those they observed in patients . in critically ill patients , the percentages of daily caloric and protein requirements met are in the order of 60 to 65% , the same as the mice in the present study . this leaves open the question of whether the changes in adipose tissue morphology are a function of critical illness or a result of malnutrition . their second comparison was between the clp mice that were fed 66% of the dietary requirement and a severely restricted group that received about 15% . however , severe malnutrition may result in a decrease in metabolism , attenuating the taxing of metabolic stores . in rodents , a similar reduction in energy expenditure is noted in severely malnourished patients in an attempt to balance energy intake and expenditure . in the present study , energy expenditures , especially the magnitude of hypermetabolism due to infection , were not measured ; thus , the full impact of nutritional intake can not be adequately assessed . the authors do provide additional data from critically ill patients reaffirming their earlier observations , and assess the effects of restricted nutritional intake . these data allow the animal data to be put into context . in the patients , the nutritional differences were the result of providing parenteral supplementation . in the patients who had increased supplementation , there appears to still be an energy deficit , especially in light of a 70% infection rate . the question that has not been addressed in the present effort is : if adequate nutrition to meet energy requirements , including the initial deficit , is provided , would the changes in adipose tissue be observed ? this would fully address the hypothesis that nutrition - independent factors evoke the morphological changes in adipose tissue during critical illness . the authors have previously posed the question of whether these changes in adipose tissue during critical illness are adaptive or protective . the present work scratches the surface with regard to alterations of adipose tissue in critical illness , but leaves open the role of adipose tissue in outcomes of patients with critical illness .

at present , various types of aesthetic restorative materials are widely used with different properties and colors . composite resins are among the tooth - colored restorative materials of choice for many dentists due to their high acceptance by patients and their ability to bond to tooth structure , their excellent esthetic properties , favorable strength , relatively low cost ( compared to ceramics ) and application in both anterior and posterior teeth [ 13 ] . the success of composite restorations highly depends on the color stability of resin in long - term . perceptible discoloration of the restorative materials may compromise restoration esthetics , which is among the most common reasons for replacement of anterior composite resin restorations [ 1,3 , 4 ] . . color change of the resin matrix itself or the matrix / fillerinterface , chemical discoloration related to the alteration or oxidation of amine catalyst , resin matrix structure and unreacted methacrylates due to incomplete polymerization comprise the intrinsic factors . extrinsic staining occurs as the result of adsorption and/or absorption of staining materials due to exposure to external sources . the degree of color change varies from patient to patient based on oral hygiene status , nutritional habits , smoking status and consumption of different beverages . numerous studies have demonstrated the effect of staining solutions such as red wine , coffee , cola and tea on composite resins . according to ardu et al , in 2010 , the degree of composite color change depends on the brand and structure of composite resin . among the staining agents , red wine caused the highest color change followed by coffee , tea , orange juice , and cola . nasoohi and ghaemi in 2013 found that color change of filtek p60 and z250 composite resins was clinically unacceptable and the degree of alteration was higher as the result of exposure to imported tea than the iranian tea . ertas et al , in 2006 reported lower stainability of filtek p60 and z250 composite resins than nano - hybrid composites and quadrant lc . red wine had the highest staining potential in comparison with coffee , tea , cola , and water , respectively . khatri and nandlal in 2010 showed that the color change of conventional composites ( tph spectrum , dentsply b1 ) was more severe than that of ceram x b1 nanocomposite especially in coffee . in a study by mahdisiar et al , in 2014 , three nanocomposites ( grandio , z350xt and herculate xrv ultra ) showed clinically unacceptable discoloration when immersed in coffee . composite resins are increasingly used in pediatric dentistry and only a few studies have evaluated the effect of commonly used beverages and medications by children particularly iron drop and multi - vitamin syrup on discoloration of composite resins . on the other hand , therefore , the purpose of this in vitro study was to assess the color change of nanofilled and flowable composite resins ( premise , kerr , orange , ca , usa ) following exposure to tea , cola , iron drop and multivitamin syrup . a2 color shades of a flowable composite resin ( premise , kerr , orange , ca , usa ) and a nanofilled composite resin ( premise , kerr , orange , ca , usa ) were used in this study ( table 1 ) . name , material type and manufacturer of the tested resin - based composites . according to curtin et al , in 2008 40 disk - shaped specimens were fabricated of each material ( 7 mm in diameter and 2 mm thick ) , using plastic molds . the molds containing composite resin were held between two glass slides , each covered with a transparent polyester strip ( mylar ; henry schein , melville , ny , usa ) , and the slides were gently pressed to remove excess material . the top and bottom surfaces of each specimen were light - cured for 40 seconds using a conventional halogen light - curing unit ( optilux 401 demetron , kerr , orange , ca , usa ) with a light intensity of 500 mw / cm . the tip of the light guide was in contact with the cover glass during the light - polymerization process . after removing the specimens from the molds , they were stored in distilled water for 24 hours at 37c for rehydration and completion of their polymerization . the top surfaces of all specimens were wet - polished with 800- , 1000 , 1500- , and 2000-grit silicon carbide papers , consecutively . four types of liquids commonly consumed by children namely tea , cola , iron drops ( ferbolin , shahredaru , tehran , iran ) and multivitamin syrup ( minadex , marfleet , england ) were tested in the present study ( table 2 ) . eight samples were randomly selected from each material and immersed in each of the four staining solutions for three hours per day at 37c over a 40-day test period . tea solution was prepared by immersing two tea bags ( ahmad tea hashtgerd , tehran , iran ) , ( 2 2 g ) into 300 ml of boiling distilled water for 10 minutes . following completion of the immersion time , the samples were rinsed under running distilled water and air - dried . samples were then stored in distilled water at 37c . staining solutions used in the study the samples were digitally photographed at baseline and after staining ( finepix s9600 , fuji , tokyo , japan ) under standardized conditions against a black background ( distance : 25 cm zoom : 1 ) . daylight was blocked out and two 6500k light sources were placed at 45-degree angles relative to the subject . the photographic procedure included a piece of gray card in the picture as a neutral reference object . color casts were eliminated and image brightness was fine tuned using a standard image - editing program ( adobe photoshop cs5 , adobe systems incorporated , san jose , ca , usa ) before the relevant color values were measured . values were recorded using the cie ( commission international de l eclairage ) l*a*b * color system . in photoshop a transformation can be done using the following formula : l*=l(pm)100/255 the a * and b * values were transformed in the same manner . the photoshop values range from 0 to 255 , and the cie l*a*b * values from 120 to + 120 . the transformation formulas are as follows : a*=[a ( pm)128]240/255b*=[b ( pm)128]240/255 where a ( pm ) and b ( pm ) are the photoshop mean values of a * and b * , respectively . the color differences or e * between the two measurements were calculated as follows : e*=[(l*)2+(a*)2+(b*)2]12 the e * values greater than or equal to 3.7 were considered as clinically - unacceptable color change that can be detected by the naked eye . two - way anova was used to evaluate the effects of material type and staining solution on color change . thus , subgroup analysis was performed with independent t - test , and bonferroni correction was also applied . a2 color shades of a flowable composite resin ( premise , kerr , orange , ca , usa ) and a nanofilled composite resin ( premise , kerr , orange , ca , usa ) were used in this study ( table 1 ) . name , material type and manufacturer of the tested resin - based composites . according to curtin et al , in 2008 40 disk - shaped specimens were fabricated of each material ( 7 mm in diameter and 2 mm thick ) , using plastic molds . the molds containing composite resin were held between two glass slides , each covered with a transparent polyester strip ( mylar ; henry schein , melville , ny , usa ) , and the slides were gently pressed to remove excess material . the top and bottom surfaces of each specimen were light - cured for 40 seconds using a conventional halogen light - curing unit ( optilux 401 demetron , kerr , orange , ca , usa ) with a light intensity of 500 mw / cm . the tip of the light guide was in contact with the cover glass during the light - polymerization process . after removing the specimens from the molds , they were stored in distilled water for 24 hours at 37c for rehydration and completion of their polymerization . the top surfaces of all specimens were wet - polished with 800- , 1000 , 1500- , and 2000-grit silicon carbide papers , consecutively . four types of liquids commonly consumed by children namely tea , cola , iron drops ( ferbolin , shahredaru , tehran , iran ) and multivitamin syrup ( minadex , marfleet , england ) were tested in the present study ( table 2 ) . eight samples were randomly selected from each material and immersed in each of the four staining solutions for three hours per day at 37c over a 40-day test period . tea solution was prepared by immersing two tea bags ( ahmad tea hashtgerd , tehran , iran ) , ( 2 2 g ) into 300 ml of boiling distilled water for 10 minutes . following completion of the immersion time , the samples were rinsed under running distilled water and air - dried . samples were then stored in distilled water at 37c . staining solutions used in the study the samples were digitally photographed at baseline and after staining ( finepix s9600 , fuji , tokyo , japan ) under standardized conditions against a black background ( distance : 25 cm zoom : 1 ) . daylight was blocked out and two 6500k light sources were placed at 45-degree angles relative to the subject . the photographic procedure included a piece of gray card in the picture as a neutral reference object . color casts were eliminated and image brightness was fine tuned using a standard image - editing program ( adobe photoshop cs5 , adobe systems incorporated , san jose , ca , usa ) before the relevant color values were measured . values were recorded using the cie ( commission international de l eclairage ) l*a*b * color system . in photoshop a transformation can be done using the following formula : l*=l(pm)100/255 the a * and b * values were transformed in the same manner . the photoshop values range from 0 to 255 , and the cie l*a*b * values from 120 to + 120 . the transformation formulas are as follows : a*=[a ( pm)128]240/255b*=[b ( pm)128]240/255 where a ( pm ) and b ( pm ) are the photoshop mean values of a * and b * , respectively . the color differences or e * between the two measurements were calculated as follows : e*=[(l*)2+(a*)2+(b*)2]12 the e * values greater than or equal to 3.7 were considered as clinically - unacceptable color change that can be detected by the naked eye . data were analyzed using spss version 16 software ( spss inc . , chicago , il , usa ) . two - way anova was used to evaluate the effects of material type and staining solution on color change . thus , subgroup analysis was performed with independent t - test , and bonferroni correction was also applied . the mean and sd values of l * , a * , b*and e * for each material are listed in table 3 . the mean values and standard deviations of l * , a * , b * and e * for the staining solutions and distilled water distilled water caused no visible color change ( e<3.7 ) . the color change values ( e * ) of tea , cola and iron drops for flowable composite and nanofilled composite were higher than 3.7 . according to the results of the two - way anova , the interactions between all materials and staining solutions were significant ( p=0.048 ) . subgroup analysis showed no significant differences between the composite resin materials for all staining solutions ( p=0.058 ) . color stability of tooth - colored restorations is of particular importance because of their aesthetics and reduction of additional cost due to frequent replacement . in addition to the above , fewer treatment sessions for restoration replacement is a top priority in pediatric dentistry . this study sought to assess the staining effect of different staining solutions on the color change of two widely used composite resin materials in primary teeth . based on a study by dozi et al , spectrophotometry and digital camera are the most reliable methods for determining the tooth color . spectrophotometer is a complex , expensive and technique - sensitive tool to determine the tooth color just in the surface . thus , we used digital radiography for color measurements as an easier and reliable tool with optimal repeatability , sensitivity and objectivity . previous studies showed that validity and reliability of digital imaging were similar or even superior to those of spectrophotometry [ 1821 ] . we chose cie lab system for measuring chromacity and recording color differences because this system is suitable for identification of small color changes and has some advantages namely repeatability , sensitivity and objectivity . in our study , color - change values ( e ) of tea , cola , and iron drops for flowable composite resin and color - change values of tea , cola , iron drops and multivitamin syrup for nanofilled composite resin were larger than 3.7 that can be detected with the naked eye . additionally , no significant difference in discoloration was noted between the tested composites . in accordance with our results , ardu et al , in 2010 demonstrated that color change of premise composite was clinically unacceptable after exposure to different staining solutions . nasoohi et al , in 2007 found no significant difference in color change between two different types of composites ( premise nanofiller and point 4 microhybrid composite ) after exposure to iranian and imported tea . in this regard , mundim et al , in 2010 reported that although color change of composite resins with high filler content was lower than that of composite resins with lower filler content , this difference was not statistically significant . mazaheri et al , in 2013 showed that there was no difference in stainability of new nanofiller composite resins in comparison with microhybrid composite resins , but type of staining solution had a significant effect on color change . patel et al , in 2004 reported that the degree of color change was related to the type of staining solution used and did not depend on the type of composite resin . surface roughness , amount and size of filler particles and the physical , chemical and mechanical characteristics of resin matrix such as water sorption , hydrophilicity and the degree of conversion are the factors affecting composite resin stainability . nanofilled composite resins are made of two different particle sizes namely nanomer ( individual and roughly spherical fillers ) and nanocluster ( loosely agglomerated collection of nanoparticles ) that have several advantages namely low polymerization shrinkage , high mechanical properties , better gloss retention , less wear and control of transparency . as stated by the manufacturer , premise trimodal is a nano - composite composed of three different types of filler components namely non - agglomerated discrete silica nanoparticles , barium glass , and pre - polymerized filler . it can be expected that nano - composites with a smaller particle size will have a smoother surface and will retain less surface stains [ 13 , 26 ] . however , in the current study , these differences were not evident , as no significant difference in discoloration was noted between the tested composites . this could be due to the similar filler particles and the fact that both composite resins were nanofilled . generally , all staining agents stained all composite resin materials ; tea produced the most severe stain and multivitamin syrup the least . the color change values in all solutions were not significantly different between the two composites ( p>0.05 ) ( table 3 ) . this finding is somehow in accord with the results of previous studies by nasoohi et al , in 2011 yousef and naga in 2012 and fontes et al , in 2009 . ertas et al , in 2006 and ardu et al , in 2010 showed that tea produced significant discoloration in some types of composite resins that is inconsistent with our results . it could be explained by the difference in the type of tested composite resins , the duration of exposure to the various staining solutions and the type of used staining solution in each study . other factors may play more important roles in the composite resin satiability than the type of staining solution . in accordance with the results of studies by ertas et al , in 2006 and zajkani et al , in 2013 , our results showed that tea caused more severe color change than cola . discoloration by tea is due to the adsorption of polar colorants onto the surface of composite resin materials , which causes more discoloration than cola . based on a study by um and ruyter in 1991 , although the low ph of cola may affect the surface integrity of the material and soften the matrix , the lack of yellow colorant in cola may be the reason why it did not cause a discoloration similar to tea . no statistically significant difference was found between the samples immersed in iron drop and multivitamin syrup . it could be due to the difference in their viscosity , presence of other compounds and iron content ( 25 mg/1cc vs. 12 mg/1cc in multivitamin syrup ) . in agreement with other studies , when composite resins were immersed in water , the color differences were imperceptible and clinically acceptable . our obtained results and those of other studies revealed that water sorption alone was not able to cause a significant color change in composite resins . although the interaction effect was significant , this effect was quantitative and not qualitative and no significant differences were found between the composite resin materials in the staining solutions . based on our study results , there was no difference in the degree of discoloration between the two types of composite resins .

various systems have been used to classify gi fistulas ; however , the three features that have been most widely used to classify fistulas are anatomical classification , output volume and etiological parameters ( 1 , 2 , 4 ) . each of these features has specific implications for the likelihood of spontaneous closure , prognosis , operative timing and non - operative care planning . these classifications are often used in combination with each other to achieve an integrated understanding of the fistula and its potential impact on the patient . external fistulas ( fig . 1 ) are pathological communications between any portion of the gastrointestinal tract and the skin , and represent the most common type of postoperative fistula . 2 ) are a connection between the gastrointestinal tract and another internal organ , the peritoneal space , retroperitoneum or the thorax ( the pleural space or the mediastinum ) ( 1 ) . classification of fistulas by output volume can be divided into high output and low output fistulas . 3 ) drain between 300 and 4,000 ml per day , and usually arise from a lesion located between the inferior third of the esophagus and the ligament of treitz . 4 ) that drain less than 100 ml per day generally arise from the ileum or colon , except in the case of intestinal malabsorption ( 4 ) . recently , the output of pancreatic and intestinal fistulas has been characterized as either high or low output according to the volume of discharge over a 24-hour period ( 1 , 2 ) . fistula closure is considered as spontaneous if no radiological or surgical intervention is required , although artificial nutrition and drug therapy may have been administered . anatomical factors that may adversely affect spontaneous fistula closure include complete disruption , a lateral ( side ) fistula , large adjacent bowel abscess , distal obstruction , a fistula tract less than 2 cm , an enteral defect larger than 1 cm , and gastric , lateral duodenal , ligament of treitz and ileal fistula sites . other factors include cancer , chemotherapy , radiation , underlying inflammatory bowel disease , uncontrolled sepsis , infected fistula fluid , hypoproteinemia , large and early leakage of the anastomosis , diabetes , corticosteroid use and renal failure ( 1 , 2 , 5 ) . it is vital to identify the source and route of the fistula tract in addition to the etiological features that may influence patient outcome such as the presence of obstructions , abscesses , or pancreatic pseudocysts . comprehensive determination of the fistula anatomy is usually obtained through radiological investigation , utilization of a plain radiograph of the abdomen , contrast studies ( barium and/or water - soluble contrast medium fistulogram ) , ultrasonography ( us ) , a computed tomography ( ct ) scan , magnetic resonance imaging ( mri ) or a radionuclide study ( 1 - 4 ) . before performing contrast studies , a plain radiograph of the abdomen helps locate surgical anastomosis clips , drains that have been left in place , and opacities or lucencies outside the alimentary tract that may indicate propagation of an abscess . in general , barium is considered the contrast medium of choice to utilize due to its ability to reveal mucosal surfaces and remain undiluted ; it can directly show the fistulous tract as well ( figs . 2 , 3 ) . however , extravasated barium may induce an acute inflammatory reaction in the thoracic or peritoneal cavity and therefore an alternative - iodinated water - soluble medium - should be used when perforation of the esophagus , stomach , small bowel or colon is suspected ( figs . 1 , 4 ) . a fistulogram is best performed by injecting contrast agent directly into the cutaneous opening in order to demonstrate the main axis and to avoid false passage into the secondary subcutaneous tracts at the time of cannulation ( fig . 1 ) . following complete visualization of the tract , further investigation to delineate associated pockets and cavities may be safely performed using angiographic catheters and guide wires under fluoroscopic guidance ( 3 ) . a water - soluble contrast agent is slowly injected and spot radiographs of several projections are obtained to delineate the anatomy of the tracts . us is portable , rapid , free of ionizing radiation , and inexpensive to perform . us is used as the first screening modality in most patients ( figs . 5 , 6 ) . however , patients who are obese , have an ileus or extensive surgical wounds may be difficult to examine . ct provides more standardized information , independent of the skill of the operator or the body habitus of the patient . the use of ct may not always visualize a fistula , though certain ct findings may suggest the need for a contrast study in order to provide a definitive diagnosis . additionally , in patients with known enteric fistulas , ct imaging may disclose other ancillary extraluminal abnormalities or complications , such as abscess formation , peritonitis , or lymphadnopathy that a contrast study alone might not reveal . most gi fistulas create abscess cavities in the intra - abdominal space , regardless of classification . the treatment of patients with significant gastrointestinal fistulas is generally a two - staged process , with initial management efforts centered on controlling bowel effluent , clearing associated abscesses and improving the nutritional status of the patient , with an ultimate therapeutic goal of closing the enteric fistula ( 6 ) . percutaneous , radiological catheter drainage is an effective and safe method for the treatment of abdominal abscesses associated with fistulas and can substitute for surgery ( fig . 7 ) . a high rate of successful percutaneous drainage has been reported ( 1 - 4 , 6 ) , with several investigators documenting a closure rate ranging from 57% to 88% following percutaneous abscess drainage . prior to the initiation of a radiological intervention , a consultation with the referring physician and a careful review of previous radiological examinations provides the interventional radiologist with a satisfactory understanding of the clinical problem . the crucial questions are the following : what is the underlying disease , what type of surgical anastomosis was performed , what is the supposed origin of the fistula , is the fistula a high - output or a low - output fistula , what does the lost fluid consist of and were surgical sump drains left and where do they lead ( 4 ) . when the decision has been made to percutaneously drain an abscess , coagulation factors and times should be checked . coverage with broad - spectrum antibiotics should be initiated before a procedure and the antibiotics altered , if appropriate , based on gram staining and cultures ; however , the risks and benefits should be weighed cautiously . percutaneous needle puncture for the treatment of fluid collection or abscess cavity can be performed under us , ct or fluoroscopic guidance ( fig . the puncture route should be meticulously planned , and the shortest pathway chosen from the cross - sectional images . there should be no intervening viscera , bowel , or vital structures such as blood vessels in the path of the needle . after diagnostic aspiration , 5 - 10 ml of contrast medium is injected to outline the cavity . under fluoroscopic observation , injected contrast medium will define the size and configuration of the abscess and any septations or will demonstrate any fistulous communication with the adjacent bowel . after final placement of the catheter , contrast medium is injected to ensure that all catheter side - holes are within the cavity and are draining properly . otherwise , side - holes within the tract can cause leakage of purulent material and the tract may not heal . occlusion of the catheter by debris can usually be resolved by saline irrigation or reaming the tube with a guidewire . ability to remove only a small portion of the irrigant indicates either 1 ) debris has partially occluded the catheter and has produced a one - way valve obstruction ; 2 ) there is a complex compartment within the cavity , and fluid flows away into a more deeply seated cavity ; 3 ) there is an internal enteric fistula ; or 4 ) the contents are semisolid , and the irrigant is spreading within the interstices of the necrotic debris ( 7 ) . the criteria for catheter removal are 1 ) volume of daily drainage < 10 ml ; 2 ) improvement in the patient symptoms ( e.g. no fever ) ; 3 ) minimal residual cavity ; and 4 ) nonpurulent drained fluid . it is critical to recognize the clinical signs of infection to determine if the catheter should be removed . usually , patients with bowel leaks or fistulas require a longer duration of catheter drainage because of persistent flow through the fistula and the relatively larger amounts of fluid present . levin tube or jejunal feeding tube insertion , under fluoroscopic guidance , is effective for decompression of bowel loops and enteral feeding . fluoroscopic or us - guided central venous catheter insertion has also been performed by interventional radiologists to provide total parenteral nutrition in high - risk patients . moreover , we have experienced many successful cases of radiological percutaneous gastrostomy or enterostomy for diversion or decompression of the gi contents . over the past decade , various alternative methods have been developed using interventional techniques such as interventional stenting ( 8 , 9 ) and interventional gluing with fibrin or histoacryl ( 10 ) . we successfully used a covered metallic stent to treat a postoperative fistula with anastomotic stricture in a patient who underwent a billroth i operation due to advanced gastric cancer ( fig . we have also successfully treated a colo - cutaneous fistula with anastomotic stricture in a patient with a descending colon segmental resection using a covered stent ( fig . when fistulous tracts have communicated with the biliary tree , transhepatic biliary drainage may be necessary . we have treated a patient with a fistulous tract located between a hepatic abscess and the jejunum . in this case , the fistulous tract was successfully treated by percutaneous drainage of the hepatic abscess ( fig . ( 10 ) recently reported the effective treatment of postoperative fistulas resistant to conservative treatment using biological fibrin glue . similarly , we successfully managed a post - traumatic jejuno - cutaneous fistula using fibrin glue ( fig . the simple blockage of a fistula tract with biological fibrin glue has a limited role ; however , in some cases , it can be an effective treatment . as long as a fistula tract is continually bathed in a combination of gastric fluid , bile , and pancreatic secretions , the tissues remain inflamed and necrotic areas can not heal . when drainage is externally controlled , a mature fibrous tract can form and the fistula will close spontaneously when the drainage tube is removed ( 3 ) . if outpatient care of the catheter is anticipated , the patient or responsible caregiver must thoroughly understand the management of the tube and need to be taught to recognize possible complications . in addition , care must be taken by members of the interventional radiology service ( both the physicians and nurses ) to explain these points , as well as how to quickly contact service members should any problem arise . in - depth consultation with the clinical service regarding drainage feasibility and overall goals prior to percutaneous treatment is essential . interventional management of gi fistulas is a valuable non - surgical therapy for seriously ill patients and is a comprehensive treatment option that includes percutaneous drainage and several other interventional procedures . however , interventional management is not easy and numerous manipulations and controls are required over many days . furthermore , not all patients will respond to interventional treatment alone . for these patients , a combination of various methods for controlling intestinal output and decreasing the amount of infection will increase the chances of a successful outcome .

the restoration of upright balance after a perturbation relies on highly automated and , to a large extent , stereotyped postural responses , involving a complex pattern of activation of upper and lower leg , trunk , shoulder , and neck muscles ( allum et al . 2002 ; bloem et al . 2000 ; carpenter et al . 2004 ; mcilroy and maki 1995 ; moore et al . 1988 . these responses typically occur at onset latencies of 100 ms . in response to a perturbation , both feet - in - place and stepping strategies can be used to recover balance , with the incidence of stepping responses becoming larger as the perturbation magnitude increases ( hsiao and robinovitch , 1998 ; mcilroy and maki 1993 ) . these responses are triggered and modulated on the basis of sensory information , with lower extremity and trunk proprioception as well as vestibular inputs as possible sources ( allum and honegger 1998 ; bloem et al . 2000 ; do et al . 1988 ) . although the onsets of automated postural responses occur before voluntary ( cognitive ) control comes into play and the characteristics of the responses are distinctly different from those of voluntary movements ( nashner and cordo 1981 ) , previous studies have shown that higher brain levels , presumably involving the cerebral cortex , can modulate postural responses by changing the activity of the pathways that are involved in their generation ( for review see jacobs and horak 2007 ) . in 1976 , nashner was the first to report that experience causes functional adaptations in response amplitudes following an unexpected change of support surface motion . subsequent studies have shown that response amplitudes depend on the predictability of perturbation magnitudes ( beckley et al . 1989 ) , restrictions on the balance recovery strategy ( feet - in - place vs. stepping ; burleigh and horak 1996 ; burleigh et al . 1994 ; mcilroy and maki 1993 ) , and the task of holding an object ( bateni et al . 2004 1981 ) . in general , these factors do not cause corresponding changes in onset latencies and activation sequences . on the other hand , both response amplitudes and onset latencies may be affected by advanced warning of the perturbation ( mawston et al . 2007 ; mcchesney et al . 1996 ) and increased postural anxiety ( carpenter et al . 2004 ) . hence , the literature indicates that changes in initial contexts generally affect response amplitudes , preserve activation sequences , and have limited influence on onset latencies . overall , this pattern of results suggests that the sensory information as generated by the induced perturbation to upright balance inevitably launches a postural response directed toward recovering or maintaining an upright stance , that can be scaled by cognitive set ( by changing the gain ) , but not fully suppressed . however , in all of these previous studies the final goal was the same in every condition , namely balance recovery . it has been postulated ( nashner and mccollum 1985 ) that in order to reduce the degrees of freedom , the cns composes complex postural responses from a combination of stereotyped synergies . when a synergy is used to recover balance in response to a perturbation , selective cancellation of single muscle activation within the synergy might not be possible . this raises the question of whether postural responses are also immutable when they are not necessarily helpful in the execution of a task . would it be possible to turn off these ( highly automated ) postural responses when functionally undesired , or can they be integrated into the motor output in a meaningful way ? the answer could provide important knowledge of the functional organization of the central nervous system in such conflicts . in order to obtain insight into this issue , an experiment would be needed to study postural responses to identical mechanical perturbations , but with distinct task demands . instruction - related modulation of long - latency stretch reflexes in response to identical single - joint mechanical perturbations has been extensively studied by instructing participants either to resist or not to resist the perturbation ( e.g. hammond 1956 ; rothwell et al . these studies demonstrated that response amplitudes of the stretched muscles ( except for the flexor pollicis longus ) were heavily modulated as a result of the instruction , characterized by divergence between instructions ( almost ) immediately after response onset . whether such instruction - related modulation also applies to automated postural responses is not known . in the present study , the effects of instruction on automated postural responses in neck , trunk , shoulder , and leg muscles were investigated when people were either instructed to recover balance after being released from an inclined standing posture , or not to recover at all and fall onto a safety mattress in the most comfortable way . it was hypothesized that instruction would have a profound influence on the whole - body postural response . two possible scenarios were anticipated . in the first scenario , the instruction of not recovering balance would result in completely different muscle onset latencies , activation sequences , and amplitudes , indicative of a separate motor program ( i.e. a set of muscle commands that are structured before a movement sequence begins ) to prepare for a safe landing and , consequently , a cancellation of the automated postural response . in the second scenario , the instruction would differentially affect response amplitudes , with no or only limited changes in onset latencies or activation patterns . this pattern of results would imply that the automated postural response could not be suppressed , but could be tailored by differential ( feedback and/or feedforward ) gain settings in order to meet specific task constraints or demands . a total of 10 healthy young adults [ 3 women , 7 men , mean age 28.3 4.3 years ( range 2237 ) , height 1.74 0.13 m , weight 68 13 kg ] participated in this study . they all provided written informed consent to participate , and the study was approved by the simon fraser university office of research ethics . the participants stood barefoot on a wooden block ( length width height : 60 38 30 cm ) located flush with a gymnasium mattress ( length width height : 480 240 30 cm ) . a tether was attached at one end to an electromagnetic brake ( warner electric model pb500 , south beloit , il ) and at the other end to a chest harness worn by the participant ( fig . the participants placed their feet at a fixed position on the wooden block and the length of the tether was adjusted such that it supported the participant in a 15 backward or leftward - inclined position ( by means of visual comparison of the lean angle with a reference line ) . for backward trials , the tether was attached to the front of the harness , whereas for leftward trials , it was attached to the right side of the harness . postural perturbations were induced by sudden release of the tether ( 90% decay time in tether force = 15 ms ) . b raw data from a typical backward balance recovery trial ( dark gray area and black , dashed lines ) and a fall trial ( light grey area and gray , solid lines ) , showing left sternocleidomastoid emg ( scl ) , anterior deltoid emg ( dal ) , rectus femoris emg ( rfl ) , right tibialis anterior ( tar ) , lateral movement of the left elbow marker , and upward movement of the right foot marker . tether release is at time = 0 ms a schematic diagram of the experimental setup ( backward perturbation position illustrated ) . b raw data from a typical backward balance recovery trial ( dark gray area and black , dashed lines ) and a fall trial ( light grey area and gray , solid lines ) , showing left sternocleidomastoid emg ( scl ) , anterior deltoid emg ( dal ) , rectus femoris emg ( rfl ) , right tibialis anterior ( tar ) , lateral movement of the left elbow marker , and upward movement of the right foot marker . tether release is at time = 0 ms prior to each trial , the participant was instructed to either recover balance [ balance recovery ( br ) trials ] or to avoid balance recovery attempts and to focus on landing safely [ fall ( f ) trials ] after the perturbation . the participants were instructed to lean into the tether and maintain their hips and knees extended , arms flexed , and hands resting on their chest . before starting the trial , the experimenter confirmed by visual inspection that the head , trunk , and legs were aligned . the participants were verbally notified of this event . after the start of the trial , the tether was released at a random time interval , varying between 1 and 7 s. release from the 15 lean angle exceeded the maximum value previously observed where young adults can recover using feet - in - place strategies ( hsiao and robinovitch 2001 ) . hence , in order to recover , it was necessary for participants to take at least one step . after release , they were also free to move their arms . in the f trials , participants were not restricted in their use of safe landing responses ( such as breaking the fall with the outstretched hands ) , except that they were not allowed to rotate around the longitudinal axis . each participant first performed the series of backward perturbations , followed by the series of leftward perturbations . muscle activities in the left and right sternocleidomastoid ( scl and scr ) , anterior deltoid ( dal and dar ) , posterior deltoid ( dpl and dpr ) , rectus abdominis ( abl and abr ) , rectus femoris ( rfl and rfr ) , and tibialis anterior ( tal and tar ) were measured through surface electromyography ( emg ; bagnoli , delsys inc . , boston , ma ) . we considered collecting data from other muscles likely involved in postural responses ( e.g. hip abductors and calf muscles ) as well . however , in order to prevent harm to both the participants and equipment , we decided not to place electrodes on potential impact sites . the emg signals were amplified , band - pass filtered ( 20450 hz ) and sampled at 960 hz . in addition , the 3d positions of skin surface markers were recorded at 240 hz with an 8-camera motion analysis system ( motion analysis inc . , santa rosa , ca ) . markers were located at the top of the head , sacrum ( l5/s1 junction ) , and bilaterally at the acromion process , lateral epicondyle of the humerus , distal end of the radius , anterior - superior - iliac spine , greater trochanter , lateral epicondyle of the femur , lateral malleolus , and fifth metatarsal . furthermore , the tether force was recorded at 960 hz from a miniature load cell ( sensotec , model 31 ) . figure 1b shows an example of emg and kinematic data ( 0200 ms after tether release ) for backward br and f trials . emg signals were full - wave rectified and low - pass filtered at 25 hz ( zero - lag , second order butterworth filter ) . for each muscle , the mean baseline and standard deviation ( sd ) in emg activity over 1 s prior to tether release were calculated . the muscle onset latency was determined by a combination of computer algorithm and visual inspection ( to ensure data quality ) on a single trial basis . onset latency for a specific muscle was defined as the time between tether release and the instant the emg amplitude for that muscle was greater than 4sd s above its baseline value , for at least 30 ms . in addition , for each trial average emg amplitudes were determined for eight bins following tether release ( one bin from 0 to 60 ms , and seven consecutive 20-ms bins from 60 to 200 ms ) . we only analyzed the first 200 ms after tether release , because we were particularly interested in whether people would be able to exert cognitive control over the initial part of the postural response , which is presumably highly automated and not generated at the level of the cerebral cortex . as it is well known that reactions at 150200 ms can certainly be cognitively controlled , emg signals after 200 ms were no longer of interest with respect to our primary research question . we also calculated several kinematic variables to determine whether they would correspond to potential differences in emg parameters . the instant of step initiation was determined as the first sample after release in which the ankle marker started moving in upward direction . the instant of initial arm abduction was determined as the first lateral movement of the elbow marker . furthermore , we quantified the initial arm movements by calculating the difference between shoulder flexion and abduction angles at tether release and at 200 ms post - release . paired t tests were used to identify differences in the instants of tether release after the start of the trial and onsets of arm abduction movements between br and f trials . analyses of variance ( anova ) for repeated measures were used with post hoc paired t tests ( with alpha levels adjusted to 0.01 to correct for repeated testing ) to detect differences in baseline activity levels and response latencies between instructions . within - subjects factors were instruction ( br and f ) , and muscle ( 12 muscles ) . anovas for repeated measures were also used for statistical analysis of response amplitudes for each muscle . within - subjects factors were instruction ( br and f ) , and bins of emg activity ( 8 bins following release ) . when significant instruction bin interaction effects were present , post hoc reverse helmert contrasts were used to determine the first bin in which the amplitudes started to deviate between instructions . in addition , anovas for repeated measures were used with post hoc paired t tests ( with alpha levels adjusted to 0.01 to correct for repeated testing ) to detect the differences in maximum amplitudes and the time of this maximum amplitude between instructions , with instruction ( br and f ) and muscle ( 12 muscles ) as within - subjects factors . finally , paired t tests were used to determine whether changes in arm positions were differentially influenced by instruction . a total of 10 healthy young adults [ 3 women , 7 men , mean age 28.3 4.3 years ( range 2237 ) , height 1.74 0.13 m , weight 68 13 kg ] participated in this study . they all provided written informed consent to participate , and the study was approved by the simon fraser university office of research ethics . the participants stood barefoot on a wooden block ( length width height : 60 38 30 cm ) located flush with a gymnasium mattress ( length width height : 480 240 30 cm ) . a tether was attached at one end to an electromagnetic brake ( warner electric model pb500 , south beloit , il ) and at the other end to a chest harness worn by the participant ( fig . the participants placed their feet at a fixed position on the wooden block and the length of the tether was adjusted such that it supported the participant in a 15 backward or leftward - inclined position ( by means of visual comparison of the lean angle with a reference line ) . for backward trials , the tether was attached to the front of the harness , whereas for leftward trials , it was attached to the right side of the harness . postural perturbations were induced by sudden release of the tether ( 90% decay time in tether force = 15 ms ) . b raw data from a typical backward balance recovery trial ( dark gray area and black , dashed lines ) and a fall trial ( light grey area and gray , solid lines ) , showing left sternocleidomastoid emg ( scl ) , anterior deltoid emg ( dal ) , rectus femoris emg ( rfl ) , right tibialis anterior ( tar ) , lateral movement of the left elbow marker , and upward movement of the right foot marker . tether release is at time = 0 ms a schematic diagram of the experimental setup ( backward perturbation position illustrated ) . b raw data from a typical backward balance recovery trial ( dark gray area and black , dashed lines ) and a fall trial ( light grey area and gray , solid lines ) , showing left sternocleidomastoid emg ( scl ) , anterior deltoid emg ( dal ) , rectus femoris emg ( rfl ) , right tibialis anterior ( tar ) , lateral movement of the left elbow marker , and upward movement of the right foot marker . tether release is at time = 0 ms prior to each trial , the participant was instructed to either recover balance [ balance recovery ( br ) trials ] or to avoid balance recovery attempts and to focus on landing safely [ fall ( f ) trials ] after the perturbation . the participants were instructed to lean into the tether and maintain their hips and knees extended , arms flexed , and hands resting on their chest . before starting the trial , the experimenter confirmed by visual inspection that the head , trunk , and legs were aligned . after the participants indicated the participants were verbally notified of this event . after the start of the trial , the tether was released at a random time interval , varying between 1 and 7 s. release from the 15 lean angle exceeded the maximum value previously observed where young adults can recover using feet - in - place strategies ( hsiao and robinovitch 2001 ) . hence , in order to recover , it was necessary for participants to take at least one step . after release , they were also free to move their arms . in the f trials , participants were not restricted in their use of safe landing responses ( such as breaking the fall with the outstretched hands ) , except that they were not allowed to rotate around the longitudinal axis . each participant first performed the series of backward perturbations , followed by the series of leftward perturbations . muscle activities in the left and right sternocleidomastoid ( scl and scr ) , anterior deltoid ( dal and dar ) , posterior deltoid ( dpl and dpr ) , rectus abdominis ( abl and abr ) , rectus femoris ( rfl and rfr ) , and tibialis anterior ( tal and tar ) were measured through surface electromyography ( emg ; bagnoli , delsys inc . , we considered collecting data from other muscles likely involved in postural responses ( e.g. hip abductors and calf muscles ) as well . however , in order to prevent harm to both the participants and equipment , we decided not to place electrodes on potential impact sites . the emg signals were amplified , band - pass filtered ( 20450 hz ) and sampled at 960 hz . in addition , the 3d positions of skin surface markers were recorded at 240 hz with an 8-camera motion analysis system ( motion analysis inc . , santa rosa , ca ) . markers were located at the top of the head , sacrum ( l5/s1 junction ) , and bilaterally at the acromion process , lateral epicondyle of the humerus , distal end of the radius , anterior - superior - iliac spine , greater trochanter , lateral epicondyle of the femur , lateral malleolus , and fifth metatarsal . furthermore , the tether force was recorded at 960 hz from a miniature load cell ( sensotec , model 31 ) . figure 1b shows an example of emg and kinematic data ( 0200 ms after tether release ) for backward br and f trials . emg signals were full - wave rectified and low - pass filtered at 25 hz ( zero - lag , second order butterworth filter ) . for each muscle , the mean baseline and standard deviation ( sd ) in emg activity over 1 s prior to tether release were calculated . the muscle onset latency was determined by a combination of computer algorithm and visual inspection ( to ensure data quality ) on a single trial basis . onset latency for a specific muscle was defined as the time between tether release and the instant the emg amplitude for that muscle was greater than 4sd s above its baseline value , for at least 30 ms . in addition , for each trial average emg amplitudes were determined for eight bins following tether release ( one bin from 0 to 60 ms , and seven consecutive 20-ms bins from 60 to 200 ms ) . we only analyzed the first 200 ms after tether release , because we were particularly interested in whether people would be able to exert cognitive control over the initial part of the postural response , which is presumably highly automated and not generated at the level of the cerebral cortex . as it is well known that reactions at 150200 ms can certainly be cognitively controlled , emg signals after 200 ms were no longer of interest with respect to our primary research question . we also calculated several kinematic variables to determine whether they would correspond to potential differences in emg parameters . the instant of step initiation was determined as the first sample after release in which the ankle marker started moving in upward direction . the instant of initial arm abduction was determined as the first lateral movement of the elbow marker . furthermore , we quantified the initial arm movements by calculating the difference between shoulder flexion and abduction angles at tether release and at 200 ms post - release . paired t tests were used to identify differences in the instants of tether release after the start of the trial and onsets of arm abduction movements between br and f trials . analyses of variance ( anova ) for repeated measures were used with post hoc paired t tests ( with alpha levels adjusted to 0.01 to correct for repeated testing ) to detect differences in baseline activity levels and response latencies between instructions . within - subjects factors were instruction ( br and f ) , and muscle ( 12 muscles ) . anovas for repeated measures were also used for statistical analysis of response amplitudes for each muscle . within - subjects factors were instruction ( br and f ) , and bins of emg activity ( 8 bins following release ) . when significant instruction bin interaction effects were present , post hoc reverse helmert contrasts were used to determine the first bin in which the amplitudes started to deviate between instructions . in addition , anovas for repeated measures were used with post hoc paired t tests ( with alpha levels adjusted to 0.01 to correct for repeated testing ) to detect the differences in maximum amplitudes and the time of this maximum amplitude between instructions , with instruction ( br and f ) and muscle ( 12 muscles ) as within - subjects factors . finally , paired t tests were used to determine whether changes in arm positions were differentially influenced by instruction . the average lean angles prior to release were indeed close to the intended 15 , as revealed by analysis of kinematic data from bilateral malleolus and acromion markers . average lean angles were 15.4 0.6 ( se ) for backward and 16.9 0.6 for leftward trials . there were no significant differences between lean angles in br and f trials in both backward and leftward perturbations ( p = 0.087 and p = 0.427 , respectively ) . analysis also did not yield significant differences in the instants of tether release relative to the start of the trial ( backward : 4.1 0.24 s vs. 4.1 0.27 s for br and f trials , respectively , p = 0.955 ; leftward 4.3 0.19 s vs. 4.0 0.23 s , p = 0.080 ) . of the total 120 br trials collected in this study , furthermore , in only 3 out of 120 f trials a recovery attempt ( step initiation ) could be observed . in the backward br trials , 9 participants stepped back with the right leg first , whereas 1 participant stepped with the left leg first . for this participant , in the statistical analysis of backward perturbations the muscles on the right side the stepping foot was the dominant foot ( i.e. the foot they use to kick a ball ) , whereas two participants used their non - dominant foot to step . foot lift in the backward trials occurred at , on average , 180 5 ( se ) ms after release . in backward f trials , most participants landed with near - simultaneous impact to the buttocks and the hands / forearms , similar to the backward landing configuration reported by hsiao and robinovitch ( 1998 ) . in leftward br trials , all participants recovered balance by stepping with the left leg first . left foot lift occurred at 237 6 ms after tether release . in leftward f trials , participants usually landed on the left knee , left hip , and on both left and right hands / forearms . in response to the perturbation , arm abduction movements were commonly observed in both br and f trials , but were earlier and more pronounced in br than in f trials . in backward trials , bilateral arm abduction movements occurred at on average 154 5 ms after release in br trials and at 172 8 ms in f trials ( p = 0.018 ) . in leftward trials , clear abduction movements movement onsets in br trials occurred at an average 159 4 ms after release and in f trials at 179 8 ms ( p = 0.009 ) . onsets of emg activity in response to the perturbation could generally be detected in all the muscles within 200 ms . in backward br trials , neck , trunk and leg muscles showed similar onsets ( 7080 ms after tether release ) , followed by dpl and dpr at 8090 ms and dal and dar at 90100 ms , on average ( fig . 2 ) . in the backward f trials , the overall mean onset latencies of the 12 muscles measured were delayed by 4.1 ms ( se 2.4 ms ) compared to the br trials [ f(1,8 ) = 5.601 , p = 0.045 , fig . 2 ] . at the level of the individual muscles , onsets were not significantly different between the instructions ( all p values > 0.045 ) . as can be seen from fig . 2 , the overall activation sequence remained similar between instructions . 2average onset latencies ( se ) in response to backward and leftward perturbations for left and right sternocleidomastoid ( scl and scr ) , anterior deltoid ( dal and dar ) , posterior deltoid ( dpl and dpr ) , rectus abdominis ( abl and abr ) , rectus femoris ( rfl and rfr ) , and tibialis anterior ( tal and tar ) . data from balance recovery trials are shown as black diamonds , and data from fall trials are shown as gray squares . * p < 0.01 average onset latencies ( se ) in response to backward and leftward perturbations for left and right sternocleidomastoid ( scl and scr ) , anterior deltoid ( dal and dar ) , posterior deltoid ( dpl and dpr ) , rectus abdominis ( abl and abr ) , rectus femoris ( rfl and rfr ) , and tibialis anterior ( tal and tar ) . data from balance recovery trials are shown as black diamonds , and data from fall trials are shown as gray squares . * p < 0.01 with respect to muscle activation sequences in leftward br trials , scl and scr were activated first at 7580 ms after tether release , followed by abl , abr , tal , tar , dpl , and dpr at 8090 ms , dal and dar at 90100 ms , and rfl and rfr at 100110 ms . overall , mean onset latencies of the 12 muscles were delayed by 9.3 ms ( se 4.8 ms ) in the f trials compared to the br trials [ f(1,8 ) = 20.373 , p = 0.002 , fig . 2 ) . the activation sequence was again generally preserved in the leftward f trials , with the exception of scr . the onset of this individual muscle was substantially delayed in f trials compared to br trials ( mean difference se , 18.6 5.1 ms , p = 0.007 ) . a less pronounced but significant delay was also observed in dar ( mean difference se , 9.7 2.8 ms , p = 0.006 ) . with respect to emg amplitudes , there were no significant differences in baseline activity levels between br and f trials [ f(1,9 ) = 2.149 , p = 0.177 ] . hence , the analysis of response amplitudes was not compromised by instruction - related baseline differences . emg amplitudes after tether release were generally higher in br than in f trials and these instruction - related differences could often be detected shortly after onset ( mostly within 40 ms ) . for backward trials , significant instruction bin interactions were found for all muscles [ values for f(7,63 ) ranging from 2.964 to 19.863 , all p values < 0.010 ] , except abl , abr , and rfr [ values for f(7,63 ) ranging from 0.433 to 1.815 , p values > 0.100 ] . post hoc contrasts revealed that emg amplitudes started to increase more steeply in br than in f trials at 6080 ms after release for scr , at 80100 ms after release for scl and tal , followed by dal , rfl , and tar at 100120 ms , and dar at 120140 ms [ values for f(1,9 ) ranging from 5.946 to 18.575 , p values between 0.002 and 0.037 ] ( fig . 3emg amplitudes of balance recovery ( br ) trials minus the amplitudes in fall ( f ) trials in response to backward and leftward perturbations . average differences ( se ) are shown as a function of time after tether release for bilateral sternocleidomastoid ( sc ) , anterior deltoid ( da ) , posterior deltoid ( dp ) , rectus abdominis ( ab ) , rectus femoris ( rf ) , and tibialis anterior ( ta ) . muscles on the left side of the body are shown as black dashed lines , muscles on the right are shown as gray solid lines . the arrows indicate at which instant emg amplitudes started to deviate significantly between br and f trials emg amplitudes of balance recovery ( br ) trials minus the amplitudes in fall ( f ) trials in response to backward and leftward perturbations . average differences ( se ) are shown as a function of time after tether release for bilateral sternocleidomastoid ( sc ) , anterior deltoid ( da ) , posterior deltoid ( dp ) , rectus abdominis ( ab ) , rectus femoris ( rf ) , and tibialis anterior ( ta ) . muscles on the left side of the body are shown as black dashed lines , muscles on the right are shown as gray solid lines . the arrows indicate at which instant emg amplitudes started to deviate significantly between br and f trials for leftward trials , analysis yielded significant instruction bin interactions for all the muscles [ values for f(7,63 ) ranging from 2.424 to 18.294 , all p values < 0.030 ] , except rfl and tar [ values for f(7,63 ) of 1.317 and 1.198 , p values of 0.257 and 0.317 , respectively ] . higher emg amplitudes were observed in br than in f trials , starting at 80100 ms for scl , scr , dal , and abr , followed by dar and dpr at 100120 ms , abl and rfr at 120140 ms , dpl at 140160 ms , and tal at 180200 ms [ values for f(1,9 ) ranging from 5.157 to 21.745 , p values between 0.001 and 0.049 ] ( fig . 3 ) . in both backward and leftward perturbations , maximum emg values ( table 1 ) were also significantly larger in br than in f trials [ f(1,9 ) = 27.430 , p = 0.001 and f(1,9 ) = 33.627 , p the time of maximum emg was not significantly different between backward br and f trials [ f(1,9 ) = 4.198 , p = 0.071 ] . in leftward trials , analysis yielded a main effect of instruction on the time at which maximum emg values were reached [ ( f(1,9 ) = 8.417 , p = 0.018 ] . post hoc paired t tests revealed that rfr reached peak activity significantly earlier in f than br trials ( p = 0.003 ) . continued high rfr activity levels in br trials probably indicate preparation for the stepping movement of the left leg . activity levels in the absence of stepping responses ( f trials ) started to decrease at 150 ms . table 1mean se of maximum emg amplitude and the instant after release at which the maximum value was reached for balance recovery ( br ) and fall ( f ) trialsbackwardleftwardbrfbrfmaximum emg ( mv)time of max . ( ms)scl217 21 * 149 5124 21138 5136 17 * 141 349 10140 11scr196 25 * 155 2124 28138 4112 23 * 151 832 9139 10dal249 48 * 160 395 14144 7286 53 * 163 4 66 20144 5dar239 50 * 152 551 9148 6106 21 148 570 32139 4108 45144 456 26144 3dpr208 50141 5120 29137 6211 51 * 147 371 21140 11abl124 42128 8139 40138 869 22 140 8 23 7113 6abr101 25131 687 15136 857 17 120 529 9112 7rfl179 36 148 7119 20128 629 5144 627 7152 4rfr93 19121 475 15134 5202 63 * 173 3 * 24 7156 4tal271 49 * 162 6180 44140 8270 48 * 167 7 179 37140 6tar322 51 * 152 5167 35131 8147 30139 4122 26139 5 * p < 0.01 mean se of maximum emg amplitude and the instant after release at which the maximum value was reached for balance recovery ( br ) and fall ( f ) trials hence , many of the muscles recorded showed significantly higher emg amplitudes in br than in f trials , with the earliest and most consistent differences between instructions being present in sternocleidomastoid . however , instruction - related differences in rectus abdominis were only present in leftward perturbations . furthermore , in both perturbation directions , emg amplitude in rectus femoris of the stance limb was greater in br than f trials . finally , instruction had differential effects on shoulder muscle emg amplitudes for the two perturbation directions . analysis of shoulder kinematics revealed that the differential effects of instruction on emg amplitudes also resulted in corresponding changes in shoulder abduction and flexion angles within 200 ms after tether release ( fig . 4 ) . in backward trials , the deltoids ( shoulder abductor muscles ) showed higher activity in br than f trials . this corresponded to increases in shoulder abduction angles in br trials that were more than twice as large compared to f trials ( left : 10.7 vs. 4.7 , se of the difference 2.1 , p = 0.018 ; right : 8.0 vs. 3.5 , se of the difference 1.5 , p = 0.014 , fig . 4average changes in flexion and abduction angles ( se ) of the left ( l ) and right ( r ) arm between tether release and 200 ms post - release for balance recovery ( diamonds ) and fall trials ( squares ) . * p < 0.05 average changes in flexion and abduction angles ( se ) of the left ( l ) and right ( r ) arm between tether release and 200 ms post - release for balance recovery ( diamonds ) and fall trials ( squares ) . * p < 0.05 in leftward trials , instruction - related differences , with larger amplitudes in br than f trials , were most pronounced in dpr ( shoulder abductor and extensor ) and dal ( shoulder abductor and flexor ) . these differences were observed in conjunction with larger shoulder extension movements of the right arm in br trials ( 8.9 vs. 4.2 , se of the difference 1.4 , p = 0.008 ) , as well as larger abduction movements of both the left and right arm ( left : 9.5 vs. 4.3 , se of the difference 1.0 , p = 0.001 ; right : 1.7 vs. 0.7 , se of the difference 0.7 , p = 0.007 , fig . was slightly adducted in f trials , as shown by the average right arm abduction angle being left of the zero - abduction line in fig . the aim of the present study was to investigate the influence of instruction on automated postural responses . participants were instructed to either recover balance or simply fall onto a gymnasium mat following a sudden postural perturbation . at the behavioral level , participants were highly successful in following these instructions , consistently exhibiting stepping movements for balance recovery in br trials , and suppressing stepping in the f trials . yet emg recordings revealed similar postural responses with onset latencies between 70130 ms in both br and f trials . these onset latencies are in line with those previously reported for forward tether release balance perturbations ( do et al . 1988 ; thelen et al . 2000 ) . the presently observed onset latencies are also in the same order of magnitude as the medium - latency responses ( 80120 ms ) reported for support surface perturbations ( allum et al . 2004 ) , despite the different nature of the perturbations . only limited instruction - related changes in onset latencies were observed , with slightly delayed responses in f trials , in combination with generally preserved activation sequences . in contrast , very pronounced and early differences were observed between br and f trials in response amplitudes , which were generally much higher in br than in f trials , but with clear differentiation between muscles and perturbation directions . hence , our results support the hypothesis that instruction influences the early - stage response to a postural perturbation by adjusting multiple gain settings , resulting in targeted scaling across muscles of response amplitudes . first , it should be pointed out that the higher response amplitudes in br compared to f trials could indeed be interpreted as an effect of instruction . methodological aspects , such as the analysis procedure , can not explain these amplitude differences . instruction had no effect on pre - release emg activity , hence the analysis of response amplitudes was not compromised by instruction - related baseline differences . furthermore , instruction - related differences in muscle response amplitudes could not be explained by a general time shift of the response , as maximum amplitudes were larger in br than in f trials and in most muscles , occurred at a similar time after onset of the perturbation . it may be argued that modulation of response amplitudes could arise from differences in fear of falling between conditions . carpenter and co - workers ( 2004 ) showed that increased levels of postural anxiety resulted in larger response amplitudes after a support surface rotation , in combination with shorter onset latencies in deltoid muscles . in the present study , fear of falling might be expected to be most pronounced in the f trials . in these trials , however , response amplitudes were reduced and anterior deltoid latencies were delayed . hence , it seems unlikely that the differences in response amplitudes can be explained by unequal levels of fear of falling between the conditions . this may reflect that , in our experiments , the anxiety associated with the task of recovering balance following sudden release of the tether ( although different in nature ) may have matched that associated with the task of falling onto the mat , which may of course not be the case during a real - life fall onto a hard surface . the finding that the two different sets of instructions resulted in modulation of response amplitudes , rather than having major effects on onset latencies and activation sequences , is similar to observations from previous studies ( bateni et al . 2004 ; burleigh and horak 1996 ; burleigh et al . 1994 ; horak et al . 1989 ; mcilroy and maki 1993 ; nashner 1976 ) . in these previous studies , a postural response was always required in order to meet the general task demands of maintaining upright balance . cognitive set resulted in the modulation of response amplitudes of specific muscles within the automated postural response ( bateni et al . 2004 ; burleigh and horak 1996 ; burleigh et al . 1994 ; horak et al . 1989 ; mcilroy and maki 1993 ; nashner 1976 ) . the present study provides an important addition to the current body of knowledge by indicating that , even when the occurrence of any postural response may not be desired , it can not be fully suppressed by instruction , as responses also occurred when people were not supposed to recover , but to fall . the apparently reflex - like generation of postural responses , however , does not seem to interfere with the goal of falling , as participants clearly succeeded following the instruction . it shows that in balance recovery , we can rely on highly automated responses , but these responses can also be effectively downregulated by the central nervous system when they are not desired . the present finding that emg amplitudes were differentially modulated across muscles has been reported previously ( bateni et al . 2004 ; burleigh et al . 1994 ; mcilroy and maki 1993 ; nashner 1976 ) and it has been suggested that this represents a goal - directed interaction ( burleigh et al . the differential effects of instruction , as observed in the present study , are in line with this idea of goal - related changes in response gains . one result to support the goal - relatedness of emg modulation was the differential modulation of rectus femoris amplitudes . the higher rectus femoris activation of the stance limb in br ( compared to f ) trials can be interpreted as a preparatory action , in order to carry the body weight on one leg to allow stepping with the other . in contrast , in f trials , no such preparation for weightbearing was needed , which explains the much larger attenuation of the rectus femoris response in the stance than in the stepping leg . the asymmetrical maximum emg amplitudes of rfl and rfr in the backward f trials , however , seem to indicate that some preparatory activity for stepping may have been present in these trials as well . as previously suggested by maki et al . ( 1993 ) , participants may have been unable to suppress the initiation of the preparatory changes in limb loading , but they still may have been able to abort the stepping reaction prior to foot - lift . furthermore , the differential modulation of shoulder muscle responses in leftward trials was presumably goal - related . in leftward trials , the early deviations between instructions of both the shoulder muscle emg and the corresponding kinematic patterns seemed to be related to positioning the arms to prepare for impact in the f trials , as participants tended to utilize the active response of impacting the ground with the outstretched hands . ( 2004 ) , who observed modulation of early deltoid responses when a handrail was available for grasping when balance was perturbed . in their study , arm movements were always directed toward the handrails , irrespective of the direction of the perturbation . it is insightful to consider the potential mechanisms underlying the slight ( and probably not functionally meaningful ) delay in onset latencies observed in f trials . this delay can not be explained by differences in preparation time to the upcoming perturbation , as the instants of tether release were not significantly different between the conditions . this does not exclude the possibility , however , that the delayed responses in f trials may be related to differences in preparation alertness . the time - critical nature of the br trials requires a high degree of readiness in order to recover balance successfully after the perturbation , whereas this high time pressure is not present in f trials . this difference in time pressure may have induced higher levels of alertness in the br trials , which is known to facilitate any response ( posner and boies 1971 ) . such an effect of alertness on postural responses has been previously reported by mcchesney et al . ( 1996 ) , who found reduced onset latencies when a pre - perturbation warning signal was provided . the present observation that the slight delay in onset latencies was not accompanied by major changes in activation sequences provides additional support for such a generic alertness - related mechanism . a limitation of the present study was that a relatively small set of muscles was sampled , because emg electrodes were not positioned on potential impact sites . as such , data could not be collected from the prime movers in response to leftward perturbations ( i.e. hip abductors ) , so it can not be completely excluded that activation patterns in these perturbations may have been changed as a result of the instruction . furthermore , forward perturbations ( with ventral impact sites in f trials ) were not conducted . although it is conceivable that the presently observed instruction - based modulation of response amplitudes would also apply to forward perturbations , this remains to be determined experimentally . this may have influenced the degree to which the reaction could be suppressed , because it allowed participants to preplan their reactions , which is different than most real - life falls or imbalance episodes . it can be expected , however , that these are the most ideal circumstances for suppression of automated postural responses in the f trials , yet they still occurred in response to the perturbation . it appears that these self - protection mechanisms are so deeply wired in our system that , even under rather optimal conditions , they can not easily be de - activated . in conclusion , the present study indicates that automated postural responses occur when balance is perturbed , irrespective of whether people are instructed to recover or not to recover balance . this suggests that the triggering of postural responses is organized in a reflex - like manner , with supra - spinal control primarily contributing to adjust these responses in a functional and goal - oriented way .

marine members of the archaeal phylum euryarchaeota are divided into three groups : marine groups ii ( mg - ii ) , iii ( mg - iii ) , and iv ( mg - iv ) . mg - ii archaea dominate the photic zone of oligotrophic oceans [ 1 , 2 ] , show seasonal variation , and comprise up to 90% of the total archaea and one - third of all microbial cells during spring blooms in the atlantic . metatranscriptomic analyses show that mg - ii archaea are among the most transcriptionally active microbial groups in the coastal pacific ocean , with transcription levels and patterns similar to those of pelagibacter ubique and sar86 [ 8 , 9 ] . despite their high abundance and transcription activity , nevertheless , mg - ii genomes have been assembled for mg - ii [ 10 , 11 ] , and the analyses suggest that mg - ii are motile , photoheterotrophic , and capable of degrading polymers such as proteins and lipids . to gain further insight into the diel activity of marine euryarchaeota in the red sea , we examined metagenomic bins containing mg - ii signatures . the contigs in these bins were retrieved from a cross assembly of microbial , viral , and transcriptomic samples collected at four time points during a single day in the gulf of aqaba in the red sea ( ena : prjeb19060 ) . manual inspection showed that one bin ( 169 ) contained a metagenome - assembled genome ( mag ) ( 156409 ) carrying hallmark viral genes including predicted major capsid protein ( mcp ) , portal protein , and large subunit of the terminase , as well as dna polymerase of the b family ( dnap ) . this contig contained overlapping terminal regions , suggesting that it represents a complete , terminally redundant viral genome . euryarchaeal dsdna viruses show diverse morphologies including spindle - shaped , icosahedral , pleomorphic , and head - tailed viruses . the latter group resembles the bacterial head - tailed phages ( order caudovirales ) , both in morphology and genome organization , and is currently classified into the families myoviridae , podoviridae , and siphoviridae , each of which includes bacterial and archaeal viruses [ 12 , 13 , 14 , 15 , 16 , 17 ] . no viruses infecting mg - ii have been isolated , and despite the increasing amount of viral metagenomic data [ 18 , 19 ] , candidate viral contigs from the mg - ii group have not been reported either . the protein sequence of the predicted mcp from mag 156409 showed significant , albeit moderate ( 33% identity ) , similarity solely to the mcps of haloarchaeal siphoviruses ( haloviruses ) ( figure 1a ) . three additional proteins encoded in mag 156409 , namely , primase ( figure 1c ) , portal protein , and prohead protease ( figure s2 ) , showed comparable levels of similarity to homologs from haloviruses , indicating a relatively distant evolutionary link . we used these protein sequences as queries in blast searches against the complete red sea assembly dataset , in an attempt to expand the repertoire of virus - related sequences . this search resulted in additional nine viral mags ( table s1 ) , all showing the same pattern of homology to haloviruses.figure 1unrooted maximum likelihood phylogenetic trees of conserved magrovirus genes(a ) major capsid protein ( mcp).(b ) dna polymerase b ( dnap).(c ) archaeo - eukaryotic primase ( aep).(d ) chaperonins ( thermosome subunit and groel).metagenome - assembled complete or nearly complete genomes ( mags ) of magrovirus from the red sea and tara oceans metagenomes are marked with red and light red circles , respectively . unrooted maximum likelihood phylogenetic trees of conserved magrovirus genes ( a ) major capsid protein ( mcp ) . metagenome - assembled complete or nearly complete genomes ( mags ) of magrovirus from the red sea and tara oceans metagenomes are marked with red and light red circles , respectively . see also figures s2 and s3 . to validate and expand our observations on red sea metagenomes , we used the same query sequences in a blast search against both the original tara oceans assembly datasets [ 20 , 21 ] and a reassembly of the raw sequences from this project ( see supplemental experimental procedures ) . this search yielded 15 additional putative viral genomes related to the red sea mags ( table s1 ) . all together , we identified 26 putative viral mags from two independent metagenomic projects ( red sea and tara oceans ) . in the phylogenetic tree of the euryarchaeal virus and provirus mcps , and their environmental homologs ( figure 1a ) , the mag proteins split into three distinct groups , two of which ( a , b ) join in a clade affiliated with the halovirus mcps , whereas the third group ( c ) forms a long branch with an uncertain affiliation . a similar phylogenetic pattern was observed for other hallmark caudoviral genes of the mags , namely prohead protease , portal protein , and large subunit of the terminase . whereas groups a and b cluster together in all these trees , the position of group c changed from tree to tree , suggesting rapid evolution . these findings , along with the fact that 11 mags are terminally redundant linear genomes of about 100 kbp in size ( table s1 ) , suggest that these mags represent a novel family of head - tailed archaeal viruses . because , as shown below , these mags appear to be strongly associated with mg - ii , we provisionally denote them magroviruses ( marine group ii viruses ) . we then compared the gene complements and genome organizations of the three groups of magroviruses in detail . although , as with many other archaeal viruses , most of the magrovirus genes encode proteins without significant similarity to any sequences in current databases , the genomes include two readily identifiable , compact gene blocks , the structural - morphogenetic module and the replicative module ( figure 2 ) . the structural module consists of the genes encoding mcp , portal protein , prohead protease , and terminase and closely resembles the corresponding genomic module of haloviruses ( figure 2 ) . the distinctive feature of magroviruses is the presence of a suit of genes for proteins involved in the genome replication ( figure 2b ) . all 26 genomes encode dnap , sliding clamp , clamp loader atpase ( replication factor c ) , archaeo - eukaryotic primase ( aep ) , replicative helicase ( mcm protein ) , rada - like atpase , single - stranded dna - binding protein ( ssb ) , and several nucleases ; all viruses except for group c also encode one or two atp - dependent dna ligases ( figure 2b ) . taken together , these proteins could comprise a nearly complete archaeal - type replisome , which is unusual among currently known viruses of archaea and bacteria , although some recently discovered bacteriophages also encode expansive suites of replication proteins . haloviruses that share the morphogenetic gene block with the magroviruses encompass a smaller complement of replicative genes ( figures 2 and s1 ) . the gene order within both the replicative and the structural modules of magroviruses is highly conserved , with limited rearrangements , largely in group c ( figure 2b ) , possibly , because operonic organization of functionally linked genes is important for virus reproduction . no auxiliary metabolic genes could be observed in magroviruses.figure 2genome organization in different groups of magroviruses and halovirusesfor each group , detailed genome schemes of the replicative gene block ( left , yellow to red ) and the structural gene block ( right , different shades of blue ) are shown . homologous genes with predicted functions are shown using color code ( see key at the bottom ) . green arrows indicate thermosome genes , and gray arrows indicate hypothetical proteins . split dnap genes from group b1 are marked with a narrow black arrow pointing to the regions between the split genes . genome organization in different groups of magroviruses and haloviruses for each group , detailed genome schemes of the replicative gene block ( left , yellow to red ) and the structural gene block ( right , different shades of blue ) are shown . homologous genes with predicted functions are shown using color code ( see key at the bottom ) . green arrows indicate thermosome genes , and gray arrows indicate hypothetical proteins . split dnap genes from group b1 are marked with a narrow black arrow pointing to the regions between the split genes . see also figure s1 and table s1 . with the exception of the dnap , aep , and ligases , the replicative proteins of magroviruses show low sequence similarity to homologs from cellular organisms that could be detected only through sensitive profile - profile searches . nevertheless , most of these proteins show signs of archaeal origin as indicated by the provenance of the closest homologs . due to the low sequence conservation , informative phylogenetic analysis was feasible only for dnap and aep . in the phylogenetic tree of dnaps , the magrovirus polymerases cluster with the halovirus dnaps [ 17 , 26 ] , and , together , these viral polymerases are related to archaeal dnapolb iii that is involved in lagging strand replication ( figure 1b ) . this gene is apparently subject to frequent horizontal transfer among archaea that is likely to be at least partially mediated by viruses [ 27 , 28 ] , so that the dnapolb iii phylogeny does not follow the archaeal evolutionary tree . groups a and c that were delineated by analysis of morphogenetic genes retain monophyly in the dnap tree ( figure 1b ) , whereas group b splits between two branches , one of which is affiliated with group a ( figure 1b ) . this discrepancy between the mcp and dnap phylogenies suggests genetic exchange among diverse magroviruses . in addition to groups a , b , and c , a putative new group ( labeled ? in figures 1b and 1d ) was observed in the dnap tree . the mags encoding this group of dnaps ( tentatively , group d ) are not represented in the red sea metagenomes but are present among scaffolds originating from the tara oceans project . group d genomes contain genes for dnap ( figure 1b ) , thermosome ( figure 1c ) , resolvase , and dna - dependent rna polymerase a , all with the highest similarity to archaeal homologs . so far no closed genomes of this group were assembled , and a morphogenetic gene block was not identified . therefore , although a connection to magroviruses is apparent , the nature of group d ( virus , provirus , mobile element , megaplasmid , or uncultured archaea ) remains to be determined . notably , seven group b magroviruses possess a split dnap gene , whereas in all other viral mags , the dnap gene is uninterrupted ( figures 2b and s3 ) . the split is located within the sequence encoding the catalytic domain , similarly to the dnap genes of the cyanophage p - ssp7 and methanobacterium thermoautotrophicum . interrupted dnap genes are also found in other euryarchaeota in which the inserts consist of post - transcriptionally excised inteins [ 30 , 31 ] . the positions of the splits in these archaeal polymerases and the group b magrovirus dnap are similar , but not identical , suggesting independent , convergent evolutionary events resulting in gene fragmentation . regardless of the exact evolutionary scenario , the split dnap is so far unique among archaeal viruses and supports the monophyly of magrovirus group b. the phylogenetic tree of the aep supports the monophyly of all three groups of magroviruses as well as the common origin of primases in magroviruses and haloviruses ; in this case , however , magrovirus groups a and c cluster with haloviruses , suggesting the possibility of gene exchange between these archaeal viruses ( figure 1c ) . except for group c , all magroviruses encode a dnaq - like exonuclease that can be implicated in proofreading during viral genome replication . unlike most of the other magrovirus genes , this nuclease shows significant sequence similarity only to bacterial homologs . thus , somewhat unexpectedly , magroviruses appear to have acquired genes not only from archaea but also from bacteria . a notable feature of magroviruses is the presence of two genes encoding distinct atp - dependent dna ligases in groups a and b , one within the replicative module and the other one , unexpectedly , embedded in the structural module ( figure 2b ) . the provenances of these ligases are different as indicated by phylogenetic analysis : the ligase encoded within the structural module represents a distinct family that along with the magrovirus proteins includes ligases from uncharacterized bacteria ; the ligase in the replicative block of group b is a typical archaeal variety , whereas the one in group a replicative block belongs to the distinct family known as thermostable ligases the structural modules of all magroviruses also contain another inserted gene that in different groups encodes distinct nucleases ( figure 2b ) . in groups a , b , and d , this is a homing endonuclease ( lagli - dadg family ) homologous to nucleases of group i self - splicing introns and inteins , which are also present in many bacteriophages . in contrast , in group c , the inserted gene encodes a homolog of the exonuclease subunit of the archaeal dna polymerase d . in addition to the conserved replicative genes , several genes implicated in replication are found in individual groups of magroviruses , e.g. , ribonucleotide reductase in group b and snf2 family helicase in group a. apart from the replicative and structural - morphogenetic proteins , 12 of the 26 magroviruses encode either a bacterial - type chaperonin groel ( groups b , b1 , and c ) or a thermosome subunit , the archaeal homolog of groel ( groups a and d ) ( figure 2b ) . unlike the replicative genes , these magrovirus proteins are highly similar to the archaeal and bacterial homologs . phylogenetic analysis confirmed the sharp split between groel and thermosome subunits ( figure 1d ) . the thermosome subunits of magroviruses group with homologs from mg - ii , which is compatible with relatively recent acquisition of these genes by the viruses . a subset of mg - ii archaea encode groel instead of the thermosome subunit , conceivably owing to displacement of the ancestral archaeal chaperonin by a bacterial homolog . although the magrovirus groel do not group with those of mg - ii in the phylogenetic tree , this could be due to the accelerated evolution in the viruses ; acquisition of this gene from mg - ii archaea remains likely . comparison of the topology of the chaperonin tree with those of the mcp , dnap , and aep trees suggests that the common ancestor of the magroviruses acquired a groel gene that was replaced by the thermosome subunit in group a. chaperonins are not encoded by any known archaeal viruses but have been detected in several bacteriophages [ 33 , 34 ] . notably , these phages do not encode the co - chaperonin groes , and groes is not required for the phage chaperonin activity . this is likely to be the case for the magrovirus chaperonins as well . given that , in magroviruses , the chaperonin genes reside in the replicative gene cluster , the chaperonins might facilitate folding of the replicative proteins and replisome assembly . mapping reads from the red sea and from the tara oceans on the 26 magrovirus genomes indicate that these viruses are globally widespread , with high abundance in the indian ocean , the red sea , and the south pacific and atlantic oceans ( figures 3a and 3b ) , and are composed of different ecotypes ( figure 3a ) . magroviruses are highly abundant in the marine environment , third only to sar11 phages and cyanophages ( figure 4 ) . surprisingly , the overall abundance of magroviruses was found to be higher than that of sar116 phages ( figure 4 ) , previously identified as the second most abundant phage group in the oceans .figure 3global abundance of magroviruses and mg - ii and/or mg - iii(a ) recruitment plots of representatives from different magrovirus groups ( a : mag 154566 , b1 : mag 154680 , b2 : mag samea2620879_23 , c : mag 155057 ) , using reads ( viral fraction ) from surface waters ( top 5 m ) from the red sea and different tara oceans stations.(b ) non - metric multidimensional scaling ( nmds ) plot of sampling sites magrovirus abundance . mg - ii abundance is represented by a gradient of white to red contour lines . a linear response between the magrovirus abundance ordination and the mg - ii abundance variable is represented by fitted contours that are equally spaced parallel lines perpendicular to the mg - ii abundance vector ( r - sq.[adj ] = 0.432 ; deviance explained = 48% ; p value = 4.82e10).region abbreviations are as follows : io , indian ocean ; rs , red sea ; so , southern ocean ; ms , mediterranean sea ; npo , north pacific ocean ; sao , south atlantic ocean ; spo , south pacific ocean.figure 4total abundance of magrovirusesthe abundance of the magrovirus reads in red sea samples and all tara oceans microbiomes and viromes is shown along with the abundances of the putative host mg - ii , marine group i crenarchaeota , marine cyanobacteria ( synechococcus and prochlorococcus ) and their phages , sar11 bacteria and their phages , and sar116 and their phages . plots for halovirus and representatives from cultured euryarchaeota are not shown as the signals were close to zero . horizontal axis : the normalized count ( genome fragments per kilobase reference sequence per million library reads [ gfpm ] ; see supplemental experimental procedures ) . vertical axis : the sampling stations . global abundance of magroviruses and mg - ii and/or mg - iii ( a ) recruitment plots of representatives from different magrovirus groups ( a : mag 154566 , b1 : mag 154680 , b2 : mag samea2620879_23 , c : mag 155057 ) , using reads ( viral fraction ) from surface waters ( top 5 m ) from the red sea and different tara oceans stations . ( b ) non - metric multidimensional scaling ( nmds ) plot of sampling sites magrovirus abundance . mg - ii abundance is represented by a gradient of white to red contour lines . a linear response between the magrovirus abundance ordination and the mg - ii abundance variable is represented by fitted contours that are equally spaced parallel lines perpendicular to the mg - ii abundance vector ( r - sq.[adj ] = 0.432 ; deviance explained = 48% ; p value = 4.82e10 ) . region abbreviations are as follows : io , indian ocean ; rs , red sea ; so , southern ocean ; ms , mediterranean sea ; npo , north pacific ocean ; sao , south atlantic ocean ; spo , south pacific ocean . total abundance of magroviruses the abundance of the magrovirus reads in red sea samples and all tara oceans microbiomes and viromes is shown along with the abundances of the putative host mg - ii , marine group i crenarchaeota , marine cyanobacteria ( synechococcus and prochlorococcus ) and their phages , sar11 bacteria and their phages , and sar116 and their phages . plots for halovirus and representatives from cultured euryarchaeota are not shown as the signals were close to zero . horizontal axis : the normalized count ( genome fragments per kilobase reference sequence per million library reads [ gfpm ] ; see supplemental experimental procedures ) . vertical axis so far , based on microscopic measurements , head - tailed viruses appeared to be the least abundant morphotype of archaeal viruses . however , our abundance estimates for magroviruses suggest that these previously uncharacterized head - tailed viruses dominate the archaeal virome in surface marine environments . in contrast to the cyanophages , and the sar11 phages , normalized counts of magroviruses are almost negligible in the microbial fraction ( > 0.2 m ) but high in the virus - enriched fraction ( < 0.2 m ) ( figure 4 ) . thus , the principal source of the magrovirus reads appear to be free virus particles rather than cell - associated viruses , proviruses or megaplasmids . a possible explanation to this observation is that mg - ii show seasonal blooming patterns . the low magrovirus signal in the microbial fraction raises interesting questions on how magrovirus virions are maintained in the marine surface waters for long periods of time . despite the abundance of mg - ii in the oceans and their apparent ecological importance , no viruses infecting these organisms have been identified so far . here , using metagenomic approaches , we describe three distinct groups of viruses associated with mg - ii . unequivocal demonstration of the virus - host relationship between magroviruses and mg - ii is currently unfeasible due to the lack of cultivable mg - ii representatives . nevertheless , several lines of evidence strongly suggest that mg - ii archaea are indeed the hosts of magroviruses . first , our abundance estimates show that mg - ii is the dominant archaeal group in the samples from which the magrovirus genomes were assembled ( figures 3a and 3b ) . second , most of the replicative genes of magroviruses and especially the viral chaperonins show clear signs of archaeal provenance , and in some cases , a specific connection with homologs from mg - ii . furthermore , group b magroviruses encode two trnas ( trna and trna ) that are most similar to the respective trnas of mg - ii archaea . some of the marine euryarchaeal fosmids deposited in genbank lack 16s rdna genes , therefore their affiliation is not resolved , and they are deposited in genbank as mg - ii / iii . in addition , some representatives of mg - iii are abundant in surface waters , therefore we can not rule out the possibility that mg - iii are the hosts of ( some ) magroviruses . the discovery of the magroviruses is part of the growing trend in virology whereby viruses are recognized solely from metagenomics sequence analysis , as recently formalized by the international committee for taxonomy of viruses . genome analysis of the magroviruses is consistent with modular evolution of viruses whereby the structural and replicative modules have distinct origins . magroviruses are unusual among viruses of bacteria and archaea in that they encode an elaborate , apparently ( almost ) self - sufficient replication apparatus . given the high abundance of both mg - ii archaea and magroviruses , the latter are likely to be important ecological agents , similar to cyanophages or archaeal viruses that apparently infect thaumarchaeota . while this paper was in revision , identification of a group of putative viruses overlapping with the magrovirus set described here has been reported independently . wrote the manuscript with contributions from all authors to data analysis , figure generation , and the final manuscript .

we suggest that our immunotherapy could be developed further to improve the treatment of osteosarcoma . the standard treatment consists of chemotherapy and surgical excision , and good results have been achieved in patients with osteosarcoma . however , additional methods have yet to be developed for treatment of patients who are resistant to the standard osteosarcoma treatment . nishida et al . reported little beneficial antitumor effects after reimplantation of frozen tumor tissue alone , including ineffective inflammatory response and nonspecific reaction to tumor cells . to improve immunotherapy for osteosarcoma , we developed a method using dendritic cells ( dcs ) to enhance tumor - specific immunoreactions because dcs are the main antigen - presenting cells initiating cell - mediated immune responses in vivo . to achieve stronger immune responses , it is important to control the immunosuppressive conditions in a tumor model . we focused on tgf- which is important in regulation of the balance of immunity in nature . tgf- is one of the most important factors in a tumor model because it suppresses cell - mediated immunity by inducing regulatory t lymphocyte ( treg ) production . tregs play an important role in maintaining the balance of immunity . in the tumor progressive state , tregs generally are activated and cell - mediated immunity is inhibited , particularly in dcs and cytotoxic t lymphocytes ( ctls ) [ 4 , 6 , 11 , 12 , 24 ] . we therefore hypothesized that an antitumor effect might be provoked if tregs are controlled , which would result in the activation of ctls and inhibition of metastatic tumor growth . the effect of combining dendritic cells and an anti - tgf- antibody on enhancing the immune response to the tumor was examined . our study focused on ( 1 ) measuring the levels of foxp3 , a marker of regulatory t cells , and cd8 ( + ) t lymphocytes inside the metastatic tumor lesion to evaluate inhibition of the accumulation of regulatory t cells and the increase in cytotoxic t lymphocytes ; ( 2 ) measuring changes in the metastatic tumor volume ; ( 3 ) counting regulatory t cells using two markers , foxp3 and cd4 , in the spleen of mice to evaluate whether inhibition of regulatory t cells was attributable to treatment with the anti - tgf- antibody ; and ( 4 ) measuring the levels of ifn- as a marker of cell - mediated immunity and il-10 as the suppressive factor of cell - mediated immunity . lm8 cells , derived from dunn osteosarcoma , were provided by the riken bioresource center ( saitama , japan ) . the cells were maintained in complete medium consisting of rpmi 1640 supplemented with 10% heat - inactivated fetal bovine serum , 100 g / ml streptomycin , and 100 u / ml penicillin . a total of 1 10 lm8 cells ( a murine osteosarcoma cell line ) was implanted hypodermically into the subcutaneous gluteal region of 60 female c3h mice that were 6 to 8 weeks old . we purchased these c3h mice from sankyo labo service corporation inc ( toyama , japan ) and housed them in a specific pathogen - free animal facility in our laboratory . 1 ) : ( 1 ) the tumor was excised 14 days after inoculation ( ex ; n = 20 ) ; ( 2 ) the tumor was excised and intraperitoneal injection of anti - tgf- antibody was performed twice per week ( ex + anti - tgf- ab ; n = 20 ) ; and ( 3 ) the tumor was excised and dcs exposed to cryotreated tumor lysates were injected twice a week into the subcutaneous contralateral gluteal region and intraperitoneal injection of anti - tgf- antibody was performed twice per week ( ex + dc[ly ] + anti - tgf- ab ; n = 20 ) . we did not observe any recurrence of the tumor at the primary site of inoculation after excision . all experiments were performed under the guidelines for animal experiments as stipulated by the oita university graduate school of medical science.fig . 1a cwe established the following three groups : ( a ) ex , ( b ) ex + anti - tgf- ab , and ( c ) ex + dc(ly ) + anti - tgf- ab . sc = subcutaneous we established the following three groups : ( a ) ex , ( b ) ex + anti - tgf- ab , and ( c ) ex + dc(ly ) + anti - tgf- ab . sc = subcutaneous bone marrow - derived dcs were generated as described by lutz and rssner with minor modifications . briefly , erythrocyte - depleted mouse bone marrow cells obtained from flushed marrow cavities ( 1 10 cells / ml ) were cultured in complete medium with 20 ng / ml recombinant mouse granulocyte macrophage colony - stimulating factor ( gmcsf ) ( peprotech ec ltd , london , uk ) in 10-cm tissue culture dishes at 37c in an atmosphere containing 50 ml co2 per l. the freeze - thawed tumor lysate was added to the dc cultures on day 6 at a ratio of five dc equivalents to one tumor cell ( ie , 5:1 ) and incubated at 37c in an atmosphere containing 50 ml co2 per l. after 24 hours of incubation , nonadherent cells including dcs were harvested by gentle pipetting . after 2 weeks of tumor inoculation , the tumor mass was resected and soaked in liquid nitrogen for 20 minutes to kill the tumor cells . we added the freeze - thawed tumor lysate to the dc cultures on day 6 at a ratio of five dc equivalents to one tumor cell ( ie , 5:1 ) . after 24 hours of incubation , nonadherent cells , including dcs , were harvested by gentle pipetting . the monoclonal antibody 1d11 ( mouse igg1 neutralizes all three isoforms of tgf- ; 5 mg / kg ) was provided by genzyme corporation ( framingham , ma , usa ) . the tgf--neutralizing activity of the 1d11 antibody was confirmed with the mink lung cell assay . the markers foxp3 and cd4 , which are expressed on the surface of tregs , were used to count the number of tregs in the spleen by fluorescence - activated cell sorting ( facs ) analysis . foxp3 is a master gene for differentiation of treg and it is a specific molecular marker of tregs as well . tregs were counted with a facs calibur flow cytometer ( becton dickinson , san jose , ca , usa ) and stained with fluorochrome - conjugated antibody ( bd pharmingen , tokyo , japan ) for the following markers : phycoerythrin ( pe)-conjugated antimouse foxp3 staining kit ( ebioscience , san diego , ca , usa ) and fluorescein isothiocyanate ( fitc)-conjugated rat antimouse cd4 ( bd pharmingen , clone : rm4 - 5 ) . data analysis was performed with cellquest software ( becton dickinson ) . tumor volumes were measured using the micro - ct apparatus ( r_mct ) which allows us to obtain high - resolution ct images in small living animals . the i - view - r ( j. morita mfg corp , kyoto , japan ) was used as the viewer , and diagnosis was made with slice images viewed in all directions . tumor volumes were estimated using the formula ( long axis short axis short axis)/6 . ifn- is increased when the cell - mediated immunity is enhanced and il-10 suppresses the cell - mediated immunity . we measured murine ifn- and il-10 release by enzyme - linked immunosorbent assay using quantikine ( r&d systems , minneapolis , mn , usa ) according to the manufacturer s protocol using an easy reader ear340 microtest plate reader ( slt labinstruments , salzburg , austria ) . immunohistochemistry was used to measure the levels of foxp3 , a marker of tregs , and cd8 , a marker of cytotoxic t lymphocytes , inside metastatic tumor lesions . lung specimens were fixed in 20% formalin and embedded in paraffin . in each case , we examined all formalin - fixed , paraffin - embedded tumor tissue blocks . five samples per mouse were cut into 4-m - thick slices . rehydrated tissue sections were incubated with primary abs against cd8 ( + ) ( santa cruz biotechnology , santa cruz , ca , usa ) and foxp3 ( abcam , cambridge , ma , usa ) diluted at 1:200 in ab diluent ( dako chemmate , kyoto , japan ) overnight at room temperature . for cd8 ( + ) staining with fitc donkey antirabbit igg and foxp3 ( + ) staining with texas red goat antirat igg ( invitrogen , carlsbad , ca , usa ) , secondary antibodies were diluted at 1:300 in ab diluent and added for 60 minutes at room temperature in the dark . digital images were taken on a biorevo microscope equipped with a confocal microscopy system ( bz-9000 ; keyence , osaka , japan ) . we determined differences among the three groups using a nonrepeated measures anova ( and the scheffe test ) . all analyses were conducted using spss 18.0 software ( spss japan inc , tokyo , japan ) . foxp3 , a marker of tregs , was substantially decreased , and cd8 ( + ) , a marker of cytotoxic t lymphocytes , was substantially increased in anti - tgf- antibody - treated groups in metastatic tumor lesions . foxp3 ( + ) cells were not recruited to the metastatic area in the anti - tgf- antibody - treated groups compared with the excision - only group ( fig . the number of cd8(+ ) t lymphocytes per unit area was greater ( p < 0.0001 ) in the mice that received dcs exposed to cryotreated tumor lysates with an intraperitoneal injection of anti - tgf- antibody ( 25.73 2.65 cells / mm ) than in the mice that received only an intraperitoneal injection of anti - tgf- antibody ( 12.08 7.61 cells / mm ) ( fig . the number of foxp3 ( + ) t lymphocytes per unit area was lower ( p < 0.0001 ) in the mice that received an intraperitoneal injection of anti - tgf- antibody ( 7.73 3.32 cells / mm ) than in the excision group ( 30.72 6.61 cells / mm ) ( fig . 2a eto evaluate cd8 ( + ) t lymphocytes and tregs in pulmonary metastasis , immunostaining was performed in ( a ) the ex group , ( b ) ex + anti - tgf- ab group , and ( c ) ex + anti - tgf- ab + dc(ly ) group . the number of cd8 ( + ) t lymphocytes ( stain , fitc donkey anti - rabbit igg ; original magnification , 200 ) was increased , and the tregs ( stain , texas red goat anti - rat igg ; original magnification , 200 ) were decreased in the combined therapy of the ex + dc(ly ) + anti - tgf- ab group . the numbers of ( d ) cd8(+ ) t lymphocytes and ( e ) tregs per unit area in the three treatment groups are shown . mice that received ex + dc(ly ) + anti - tgf- ab group showed the highest level of cd8 ( + ) t lymphocytes and the lowest level of tregs . to evaluate cd8 ( + ) t lymphocytes and tregs in pulmonary metastasis , immunostaining was performed in ( a ) the ex group , ( b ) ex + anti - tgf- ab group , and ( c ) ex + anti - tgf- ab + dc(ly ) group . the number of cd8 ( + ) t lymphocytes ( stain , fitc donkey anti - rabbit igg ; original magnification , 200 ) was increased , and the tregs ( stain , texas red goat anti - rat igg ; original magnification , 200 ) were decreased in the combined therapy of the ex + dc(ly ) + anti - tgf- ab group . the numbers of ( d ) cd8(+ ) t lymphocytes and ( e ) tregs per unit area in the three treatment groups are shown . mice that received ex + dc(ly ) + anti - tgf- ab group showed the highest level of cd8 ( + ) t lymphocytes and the lowest level of tregs . error bars represent sd . the change in metastatic tumor volume was smaller in the anti - tgf- antibody and dc - treated group . the volume of the metastatic lesion ( p < 0.0001 ) in the mice that received dcs exposed to cryotreated tumor lysates with intraperitoneal injection of anti - tgf- antibody was significantly smaller ( 388.49 34.48 mm ) than in the group that received only intraperitoneal injection of anti - tgf- antibody ( 1104.53 104.88 mm ) ( fig . 3).fig . 3the volume of lung metastases in the three treatment groups is shown . the anti - tgf- antibody was able to markedly reduce the cell population of the tregs whose two markers are cd4 ( + ) and foxp3 ( + ) in the spleen . the group that received an intraperitoneal injection of anti - tgf- antibody showed a marked decrease in the percentage of cd4 ( + ) foxp3 ( + ) cells compared with the excision group ( fig . 4a cthe ratio of cd4 ( + ) and foxp3 ( + ) t cells in the spleen at day 42 after tumor inoculation was measured using flow cytometry in ( a ) the ex group , ( b ) ex + anti - tgf- ab group , and ( c ) ex + anti - tgf- ab + dc(ly ) group . cd4 ( + ) foxp3 ( + ) t cells decreased in the anti - tgf- antibody groups . the ratio of cd4 ( + ) and foxp3 ( + ) t cells in the spleen at day 42 after tumor inoculation was measured using flow cytometry in ( a ) the ex group , ( b ) ex + anti - tgf- ab group , and ( c ) ex + anti - tgf- ab + dc(ly ) group . cd4 ( + ) foxp3 ( + ) t cells decreased in the anti - tgf- antibody groups . an increase in serum ifn- level and a decrease in il-10 level mean that cell - mediated immunity was enhanced in the dc and anti - tgf- antibody - treated group . mice treated with dcs exposed to cryotreated tumor lysates with intraperitoneal injection of anti - tgf- antibody showed higher serum ifn- levels ( 221.81 16.85 0.0001 ) than the group that received only intraperitoneal injection of anti - tgf- antibody ( 130.01 16.07 0.0001 ) in the mice that received dcs exposed to cryotreated tumor lysates with intraperitoneal injection of anti - tgf- antibody ( 20.33 9.05 pg / ml ) than in the group that received only intraperitoneal injection of anti - tgf- antibody ( 103.73 18.37 b(a ) mice that received ex + anti - tgf- ab + ( b ) mice that received ex + anti - tgf- ab + dc(ly ) had the lowest levels . ( a ) mice that received ex + anti - tgf- ab + dc(ly ) had the highest ifn- levels . ( b ) mice that received ex + anti - tgf- ab + dc(ly ) had the lowest levels . although adjuvant therapy is the most effective treatment strategy for osteosarcoma , new treatment methods for metastatic osteosarcoma should be developed . in the 1970s , immunotherapy for osteosarcoma was reported by marcove et al . , southam et al . , marsh et al . , and gainor et al . . in addition , several immunotherapies have been attempted as new methods to overcome progressive cancer [ 7 , 17 , 18 ] ( table 1 ) . we focused on the inability to control the factors that inhibit the attacker cells such as dcs and cd8(+ ) t - lymphocytes as a major cause of insufficient antitumor effects . our purposes were to ( 1 ) evaluate the levels of foxp3and cd8 ( + ) t lymphocytes inside the metastatic tumor lesion ; ( 2 ) determine the changes in metastatic tumor volume ; ( 3 ) count the regulatory t cells in the spleen of mice to evaluate inhibition of regulatory t cells ; and ( 4 ) measure the levels of ifn- and il-10.table 1immunotherapeutic trials in malignant tumorsstudytumorimmune interventionrouteimmunologic responsecommentsmarcove et al . osteogenic sarcomairradiated autogenous vaccinescnc , pdactivate immunization using homogenized tumor cell is encouragingsoutham et al . osteogenic sarcomairradiated autogenous vaccinescnc , dodthe relatively high proportion of patients who apparently are curedmarsh et al . osteosarcomalymphocyte proliferation assayin vitroin vitrolpa may be useful in monitoring clinical progress of the diseasesoleimani et al . renal cell carcinomadcs and tumor lysatescnc , pddepletion of regulatory t cells should be attained by combination of conventional cancer treatment and cancer vaccine immunotherapyrosenberg et al . prostate cancerdcsscnc , pddcs can directly mediate tumor - directed cytotoxicitycurrent studyosteosarcomadcs and tgf-sc , ipprcombining dcs with anti - tgf- antibody resulted in enhanced antitumor effectsdcs = dendritic cells ; sc = subcutaneous ; nc = no change ; dod = dead of disease ; pr = partial response ; iv = intravenous ; ip = intraperitoneal ; cr = complete response ; pd = progressive disease ; lpa = lymphocyte proliferation assay ; tils = tumor - infiltrating lymphocytes . immunotherapeutic trials in malignant tumors dcs = dendritic cells ; sc = subcutaneous ; nc = no change ; dod = dead of disease ; pr = partial response ; iv = intravenous ; ip = intraperitoneal ; cr = complete response ; pd = progressive disease ; lpa = lymphocyte proliferation assay ; tils = tumor - infiltrating lymphocytes . first , we used mice with an identical genetic makeup and dcs from a different ( albeit genetically identical ) mouse in transfer experiments . clinical applications would require the use of dcs derived from the same individual , which was not possible in our mouse model . second , because we used mouse osteosarcoma cells and dcs , we were unable to test the induction of human osteosarcoma and the data may not reflect the responses of the human immune system . further studies are required to confirm these observations across a wide range of models for clinical application . anti - tgf- antibody inhibited the accumulation of tregs and induced the infiltration of cd8 t cells inside the metastatic lesion . tgf- signaling in cd4 ( + ) cd25(+ ) treg is required for their immunosuppressive capacity . we showed blockage of tgf- led to the inhibition of foxp3 ( + ) t cells inside the tumor tissues . however , the inhibition of tgf- and tregs enhances antitumor immunity and the risk of autoimmune diseases developing or cytokine storm . establishing the adequate dosage of antibody and careful monitoring to prevent the occurrence of severe adverse effects are necessary for clinical application of this antitumor therapy . the group treated with the combination of dcs and anti - tgf- antibody also showed suppression of the size of lung metastases . this is consistent with our results that metastatic lesions were substantially inhibited in the combined therapy group , suggesting the control of the immunosuppressor factors may facilitate the activity of ctls in the tumor microenvironment . inhibition of tregs accumulation in the spleen can produce enhancement of the systemic cell - mediated immunity through activation of the dcs or cytotoxic t lymphocytes . we believe that this could suggest the reduction of tregs and proliferation of cd8 ( + ) t lymphocytes inside lung metastatic lesions ( fig . 2 ) . blockage of tgf- induced activation of cell - mediated immunity by increasing serum ifn and reducing il-10 . tgf- causes potent immunosuppression mediated by cytokines from tumor cells , and tgf- blockage may be useful for enhancing cancer therapy or vaccine efficacy [ 10 , 21 , 23 , 25 ] . our results for the serum cytokine levels suggest that the anti - tgf- antibody not only inhibited the accumulation of regulatory t lymphocytes as immunosuppressors but also enhanced cell - mediated immunity . currently , chemotherapy for human osteosarcoma is the standard treatment , and it should be administered before immunotherapy . the effectiveness of the anti - tgf- antibody should be evaluated in combination with current chemotherapies for treatment of human osteosarcoma . biswas et al . reported that mice treated with the chemotherapy drug doxorubicin showed elevated levels of tge- and tumor spread . therefore , the combination of chemotherapy with anti - tgf- antibody could be effective for treatment of patients with residual tumors or distant metastasis after chemotherapy . these issues will be addressed in future studies to enable clinical application of our therapy for treatment of human osteosarcoma and to facilitate further research efforts . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

there are approximately 1.5 billion people in developing countries without access to electricity and 2.5 billion people who are dependent on traditional biomass fuels for cooking and heating . in jamaica , the island 's electricity supply industry is the jamaica public service company ( jpsco ) which was established in 1923 and currently have a customer - base of 582,000 ( 92% ) - operated under the auspice of marubeni corporation ( 80% shares ) and the government of jamaica ( 20% share ) . the electricity company , which is vertically integrated , owns and operates 28 generating plants , 54 substations , and approximately 14,000 kilometres of distribution and transmission lines . while it may be argued that modern forms of energy can liberate millions of women and children from the burden of gathering water and fuel - wood , the fact is that alternate use of lighting result in approximately two million premature deaths per year , worldwide and more than five million ( mostly children ) die from illnesses caused from drinking poor quality water . some 1.1 billion people ( 18% ) of the world 's population lacked access to potable water by the end of 2004 . in jamaica , the national water commission ( nwc ) , jamaica 's state - owned water supply industry , reports that over 770 billion gallons of potable water was produced . in 2005 , the nwc produced approximately 296,454.1 megalitres of potable water . this was 5.8 percent higher than production for 2004 and reflected increased production in the 14 parishes of jamaica , hence overall access to potable water represents 86.2% . this research investigates the extent to which alternate electrification and limited access to potable water impacts the health of jamaicans within the age range of 35 - 49 and greater than 50 . the former was collected via the use of a structured 24-item interview schedule , while the latter via intense literature review . the sample size represented 150 respondents ( 75 male and 75 female ) from the 14 parishes of jamaica . the respondents involved in the sample were adults , who appeared to be of sound minds and who represented the various levels of education . the respondents were interviewed face - to - face from various locations : the food markets , on the streets ( including people travelling from the field , rivers and standpipes with loads on their heads ) , at home , construction sites and business places . in an effort to determine the number of persons to be interviewed , each parish population was divided by the island 's population of 2,599,334 and then multiplied by 150 . the number of cases interviewed per parish is noted in table 1 . distribution of the sample by parish the objective of the interview schedule was to capture the relevant information that would help to determine the extent to which alternate lighting / electricity and limited access to potable water as public goods , impacts on health and well - being of jamaicans . the format of the instrument reflected three overarching considerations , namely : demographic characteristics ( age , gender , educational attainment , area of residence , occupation ) , socio - economic status ( public utility charges and consumers ability to pay , alternate use of public utilities ) , medical implications ( head - loading , back injury , nutritional defects and treatment ) . table 2 shows the demographic characteristics of the respondents ( 35 - 50 ) , majority of whom represented the 40 - 49 age range ; attained education mainly at secondary ( male = 33% ) and tertiary ( female 49% ) levels . majority of the male respondents were farmers ( 40% ) and trade workers ( 31% ) , while their female counterparts , were mainly administrators / clerical ( 39% ) and also farmers ( 21% ) . more female ( 57% ) than male ( 44% ) faced economic constraints , hence non - access to electricity . this is followed by grid network distance ( male = 39% , female = 29% ) and include communities such as adeous valley in st . in addition , some of the respondents refused to maintain electricity service ( male = 17% , female = 13% ) because of negative experience ( frequent billing anomaly , wrongful disconnection , unsatisfactory customer care ) with the electricity company , as well as because of not possessing the modern amenity that would require the use of such service ( indicated in table 3 ) . despite the limited access to electricity , table 4 shows significant improvement in lighting over the period 1994 - 2007 . to be more specific , the most common form of lighting in jamaica is electricity , followed by a common substitute : kerosene the use of kerosene has shown significant decline prior to and over the period of privatization , with its recent statistics showing 6.3 percent in 2007 , compared to 11.3 percent at the start of privatization ( 2001 ) . other lighting sources ( including candles ) the aforementioned evidenced that owing to an increase of electricity service throughout the island since privatization , the use of kerosene decreases . demographic characteristics limited access , alternate use and head loading source of electricity per jamaican population on the point of potable water , this is supplied by the national water commission of jamaica . like electricity , economic constraint was the highest deterrent to accessing potable water , moreover for female - headed households ( 45% ) than male - headed households ( 36% ) . this is followed by limited public standpipes ( male = 37% , female = 37% ) and hilly terrain ( male = 27% , female = 17% ) - refer to table 3 . table 4 also shows alternate use of public utilities ( electricity and water ) and load carrying . public standpipe ( male = 37% , female = 27% ) and spring water ( male = 25% , female 26% ) were most likely to be consumed by the respondents who were not in contractual arrangement with the national water commission . these individuals ( male = 71% and female = 68% ) usually transport heavy pans of water on their heads . on the other hand , females were more likely to do their laundry at a nearby river and transport same on their heads ( 17% ) . thomas ( rowland 's field ) , portland ( bibrook , skibo and belleview in portland ) , st . catherine ( mountain path ) , trelawny ( freeman 's hall , stettin , wirefense , albert town and joe hut ) , westmoreland ( haddoc ) , st . the lewis store community obtains water in their pipes on average thrice per week , while residents of heywood hall get water once in a while . the residents of the above - named communities in trelawny have to walk many miles to obtain water from standpipes , the quashi river and springs . in addition , the transporting of heavy loads of firewood and pans of water and laundry on one 's head , results in the respondents reporting injury to their backs / spine ( male = 67% , female = 38% ) . access to potable water through the national water commission ( jamaica 's state - owned water supply industry ) has seen improvements ( but with slight fluctuation ) over the period 1993 - 2007 . although such improvement in terms of residents accessing potable water in their home pipes , the use of public standpipes decreases , but shows no significant difference in access to rain , river , spring and pond water . in essence , persons are more likely to access potable water via home pipes and public standpipes ( according to table 5 ) . source of water per jamaican population with regard to firewood , this was mostly used for cooking outdoor , as well as indoor ( especially during rainy season ) and was mostly utilized by female ( 15% ) than male ( 12% ) , as the respondents possessed neither electricity nor gas stove . such firewood is also frequently transported via one 's head , mainly by male ( 29% ) than female ( 15% ) and was common among residents of adeous valley in st . elizabeth , rosewell in clarendon , and wirefence , all - sides and crown land in trelawny . in addition , all the respondents used kerosene lamps as an alternate form of lighting . this is owing to economic constraint , grid network distance and the non - desire to absorb electricity service from the jamaica public service company . inhalation of kerosene from the lamp resulted in lamp oil poisoning ( male = 12% , female = 19% ) . during the interview , the respondents ( kerosene lamp users ) complained of having encountered symptoms such as throat swelling , vision problem , breathing restrictions , dizziness , euphoria and gastrointestinal problems ( vomiting , blood in stool and abdominal pain ) . in addition , the use of firewood resulted in asythma and sinusitis , obstructive pulmonary disease , including emphysema and bronchitis ; significantly more in the female ( 43% ) respondents than their male counterparts(21%).the absence of electricity often results in some of the respondents storing their meat via the use of large quantity of salt ( instead of a refrigerator ) for preservation . this resulted in nutritional defects such as paroxysmal abdominal pain ( colic ) and diarrhea ( male = 91% , female similarly , the consumption of non - potable / contaminated water caused gastroenteristis among the respondents ( female = 89% , male = 61% ) , who were more likely to use home remedies ( cola acumulata , momordica charantia and the first urine in the morning ) rather than consult with a medical practitioner . lack of or limited access to electricity as public good , has the propensity to affect both health and economic well - being of individuals . take for instance where kerosene lamp is used as an alternate form of lighting , this resulted in kerosene oil poisoning . consistent with this study , one school of thought discussed the harmful effects in breathing kerosene , especially if it is used for the purpose of lamp , cooking or heating . it was noted that there can be effects on the airways and lungs ; eyes , ear , nose and throat ; gastrointestinal ; heart and blood ; nervous system ; and skin . such effects are as a result of hydrocarbons , substances that contain only hydrogen and carbon . on the point of economic well - being , poor households often pay 10 or 20 times the price paid by connected households with regular service , thus resulting in benefits reaped by those households whenever services are expanded . households with electricity pay less than us$0.10 per kilowatt hour ( kwh ) to light up their homes , while those without , tends to rely on candles that cost the equivalent of us$5 per kilowatt hour . in port - au - prince , haiti , households with piped water connections pay us$1.00 per cubic meter , but those without pay us$10 per cubic meter to obtain water from private vendors . the former costs us$2.20 per 750 millilitre , while the latter cost us$0.30 ( including general consumption tax of 16.5% ) per candle . on average one household would spend us$18.36 per month ( estimating that 750ml x 2 are used per week ) for kerosene oil and us$25.23 for candles ( estimating that 3 candles are used per day ) . like the case of guatamala , the use of alternate lighting is more expensive than the average electricity charges ( us$0.27 per kwh in jamaica ) . the aforementioned costs could impact on the poor , hence the counter argument against the neoclassicalist and the neoliberalist ( who argues that privatization lowers prices and that higher electricity charges comes with privatization ) . this is reflected where there is a steady increase in electricity charges since the privatization of the jamaica public service company . charges for electricity went from us$0.14/kwh at beginning of privatization in 2001 to us$0.27 per kwh in 2009 . prices and grid network proximity are two main drivers of electrification . with the absence of modern form of lighting , households are left with no choice but to seek other forms of alternative . however , it should be realized that the jamaica public service company would not reap economies of scale or economies of scope if it were to effectively employ universal access . this is because each kilometer of wires costs over us$11,236.00 , and thus poses a challenge to equitable access especially in the case of distance from grid network . the rural electrification programme , which is an initiative of the government of jamaica ( since 1975 ) , provides power on a long - term payment loan basis , where rural houses are being wired and the customers make a deposit of 10% of the total sum of us$168.54 , being the initial payment . the remaining sum is required to be paid on a monthly basis , as part of the electricity bill , which is sent from the jamaica public service company . it is imperative to note therefore that the rep contributes 12% access to electrification , hence 92% overall access to the overall customer base ( 582,000 ) of the jamaica public service company . however , where the remaining 8% lacks access , this could be embraced by the views of the world bank which states that where the supply of electricity is insufficient and unsustainable , this constitutes energy / resource poverty , and thus affects approximately 40 percent of people in the developing world . such insufficiency and unsustainability are also driven by a lack of capacity to meet electricity cost . consonant with the findings from the 1992 united nations conference on environment and development , the earth summit shows that females suffer more from a lack of electricity than their male counterparts ( especially in terms of obstructive pulmonary disease and nutritional defects ) . the views of karlsson and mcdade are that many women spent long hours gathering fuel over long distances , hence their vulnerability to the impacts of deforestation , desertification and ecosystem disruption , incompletely burnt particles from indoor fires , and pollutants such as carbon monoxide , benzene and formaldehyde . the burning of wood can cause the health of women and children to be affected because wood smoke contains gasses , fine liquid and solid particles that when breathed in can be trapped in the lungs . this ( according to the authors ) , could result in lung infections , breathing problems at various levels , runny nose , eye and throat irritations , weaker immune system , headaches , coughs , bronchitis and asthma attacks . as a matter of fact , persons with existing lung diseases such as chronic obstructive pulmonary disease ( including bronchitis and emphysema ) and asthma are at higher risk of developing breathing problems from wood smoke exposure . the literature specifically states that women and children suffer from higher levels of lung and eye disease than men because they spend more time inside the house and are therefore more likely to be exposed to indoor air pollutants such as soot released into the air when biomass fuels ( for example , wood , charcoal , dung , crop residue ) are burned for cooking and heating . the world bank has classified indoor air pollution in developing countries among the four most critical global environmental problems . this study shows that the absence of potable water to individual households also results in persons having to consume water from river , spring , standpipes , thus the propensity for gastroenteristis or nutritional defect . this is because ( according to the literature ) , contaminated water may contain harmful micro - organisms , such as viruses and bacteria such as salmonella or campylobacter and gastro - intestinal parasites such as giardia or cryptosporidium . these harmful micro - organisms , known as pathogens , are not visible to the naked eye and may even be present in clear water . drinking water containing these microorganisms can cause severe gastroenteritis , possibly lasting for several weeks . infants , the elderly and people with suppressed immune systems are more likely to be affected . in continuing on the point of nutritional defects , this study shows that the respondents without electricity would store meat by use of salt , owing to the absence of refrigeration to facilitate proper storage . the respondents reported that where such perishable product becomes contaminated , they would dispose of the contaminated area and consume the remaining portion . this sometimes results in paroxysmal abdominal pain ( colic ) or what they referred to as these respondents applied to themselves as treatment ( home remedies ) , the ingesting of momordica charantia ( cerassee ) and cola acumulata ( bissy ) , as well as ingesting a teaspoon of their first urine in the morning , while others consulted with medical professionals . it should be noted that food poisoning usually take place when the rate of entry of detoxification into the blood stream exceeds the rate of detoxification of the toxic liver . cola acumulata slows down the rate of absorption of the contaminated product in the blood stream and gives the liver more time to detoxify . the reason why bissy slows down the system is because it is high in caffeine , which takes the body out of a digestive mode . with regard to the first urine ( highest in acidic content ) , this relaxes the vasos spasm of the intestine , hence relieves the respondent 's discomfort from colic or diarrhea . such public health concern brings home the point that there is a correlation between electricity privatization and the probability of a household owning a refrigerator . this finding was as a result of a 30,000 household survey which was conducted by the ministry of health , jamaica . the author postulated that , by reducing the frequency and duration of electrical power interruptions , privatization may have an impact on the likelihood of food poisoning and the lowering child mortality rates caused by food poisoning . in view of head - loading ( that is the transporting of heavy firewood , pans of water and laundry on one 's head ) , the literature shows that this could impact negatively on the cervical spine , causing a degeneration or annular tear . in the case of this study , more male ( 67% ) than female ( 38% ) experienced back / spinal injury from head - loading . the spinal cord is made up of many small bones called vertebrae and consists of five regions : cervical , thoracic , lumbar , sacrum , and coccyx . the vertebrae are separated and cushioned by intervertebral discs , which act as shock absorbers between the vertebrae , hence help absorb the shock of any unusual load ( apart from the weight of the head and upper body ) or sudden downward pressure . in a study of the cervical spine of african head - bearers , it is revealed that load bearing on the head , produced radiographic degenerative signs , much stiffness in the neck and pain . it is further noted that head - loading imposes a considerable amount of strain to the axial skeleton . the cervical spine , being the most cranial and mobile part of the vertebral column , may be susceptible to spondylosis or disc degeneration in head - loading . it also creates a shift in the degeneration from the fifth intervertebral disc space to higher levels and alters the pattern of degenerative changes of the cervical spine . consonant with this argument , the literature also points out that the axial strain of load - carrying on the head exacerbates degenerative change in the cervical spine . the findings of this study reveal that where electricity and potable water are limited or absent and persons resort to alternative means such as the use of kerosene lamp , firewood , river , spring , etcetera , these tend to result in medical implications such as euphoria , vision problems , asthma , sinusitis , obstructive pulmonary disease , paroxysmal abdominal pain ( colic ) , diarrhea , gastroenteritis inter alia . similarly , where wood and water are being transported via head - loading , this also has health repercussions such as back / spinal injury . it means therefore , that alternate electrification and non - potable water have a negative impact on the health and well - being of individuals in jamaica .

in july 2008 , the joint united nations programme on hiv / aids ( unaids ) reported that by december 2007 , there were an estimated 33.0 million people globally living with hiv infection . it also stated that the number of cases of new hiv infections had decreased from 3.0 million in 2001 to 2.7 million in 2007 . the report showed that 2.0 million people died globally due to aids - related disease in 2007 , compared with an estimated 1.7 million aids - related deaths in 2001 . the report further stated that although the percentage of people who are living with hiv appeared to have stabilized , the overall number of people living with hiv infection had steadily increased , as new hiv infections occur on a yearly basis . according to the unaids report , southern africa continues to bear a disproportionate burden of hiv infection , with 35% of hiv infection and 38% of aids - related deaths reported in southern africa in 2007 . in total , sub saharan africa is reported to be home to 67% of all people living with hiv.1 as the hiv / aids pandemic continues to expand , especially in developing countries , the moral obligation to provide safe and efficient antiretroviral treatment has become apparent to both national and international health - care communities . standard antiretroviral therapy ( art ) consists of the use of at least three antiretroviral ( arv ) drugs to suppress maximally the hiv and stop the progression of hiv disease . there have been significant reductions seen in the rates of death and suffering following the use of a potent arv regimen . the world health organization ( who ) and unaids estimate that at least 14.6 million people were in need of antiretroviral therapy in 2009 . as of the end of 2009 , 5.25 million people had access to antiretroviral therapy in low - and middle - income countries . access to arv drugs is increasing among people living with hiv and aids in developing countries due to local , national , and international efforts.2 potent combination art , mainly consisting of three or more arv drugs , has greatly improved the health and survival rates of hiv - infected patients in areas of the world with access to arvs . more than 20 individual arvs in six classes are available in the united states , in addition to several fixed - dose combination preparations . these can be combined to construct a number of effective regimens for initial and subsequent therapy . although art has its limitations , it saves lives and improves immune system function , reduces the risk of many hiv - related and non - aids complications , and reduces the risk of hiv transmission . increasingly , several lines of evidence point to the benefit of art even for patients with high cd4 counts.3 the mortality and morbidity benefits of art are obvious in patients with relatively advanced immune suppression or with symptoms related to hiv infection . for asymptomatic patients with higher cd4 counts ( eg , > 350 cells / mm ) , the question of when to initiate art remains an area of research and debate . however , it is known that there is a spectrum of risk for adverse outcomes that increases as the cd4 count declines . in persons with cd4 counts of < 200 cells/l , effective art dramatically decreases morbidity and mortality . for persons with cd4 counts of 200350 cells / mm , data from randomized controlled studies as well as cohort studies also demonstrate a reduction in both aids and non - aids events among those who start art . a variety of data from observational cohort studies show a reduction in death as well as in aids and non - aids related complications among persons who start art with cd4 counts of > 350 cells/l rather than < 350 cells / mm.4,5 arvs have been shown to reduce significantly the rate of replication of hiv in the body of an hiv - infected person . although art does not completely destroy the virus and can not cure the infection / disease , it can however greatly decrease the viral load and significantly slow the progression of the disease , thereby increasing life expectancy and improving the quality of life of people living with hiv and aids . art is not required by all people living with hiv and aids at all stages of hiv infection , but art is recommended when the cd4 is less than 350 cells / mm . six classes of antiretroviral agents currently exist : nucleoside / nucleotide reverse - transcriptase inhibitors ( nrtis ) , nonnucleoside reverse transcriptase inhibitors ( nnrtis ) , protease inhibitors ( pis ) , integrase inhibitors , fusion inhibitors , and chemokine receptor antagonists . each class of art targets a different step in the viral life cycle as the virus infects a cd4 t lymphocyte or other target cells . the use of these agents in clinical practice is dictated by their ease or complexity of use , effect profile , efficacy based on clinical evidence , practice guidelines , and clinician preference . although nrtis have been found to be less potent against hiv than nnrtis and pis , the nrtis have had a principal role in arv treatment , remain part of the current standard of care , and have been shown to exhibit activity against hiv-1 and hiv-2.3,6,7 when hiv infects a cell , the viral rna is converted into viral dna and is then copied into the host cell s dna by an enzyme called reverse transcriptase . the protease enzyme assembles the copied viral genetic material into new viruses , after which they are released from the cell to infect other cells.3,6 the first class of arv reverse - transcriptase inhibitors operates early in the hiv life cycle to stop viral replication following hiv infection . usually , nnrtis bind to the reverse transcriptase enzyme and prevent the hiv rna from converting to dna , thereby preventing it from being copied into the cell s dna , while the nrtis incorporate into the viral dna and prevent it from producing copies of the virus . nrtis interrupt the hiv replication cycle via competitive inhibition of hiv reverse transcriptase and termination of the dna chain.8 reverse transcriptase is an hiv - specific dna polymerase that allows hiv rna to be transcribed into single - strand and ultimately double - strand proviral dna and incorporated into the host - cell genome . it is known that proviral dna chain elongation is necessary before genome incorporation can occur and is accomplished by the addition of purine and pyrimidine nucleosides to the 3 end of the growing chain . nrtis are structurally similar to the dna nucleoside bases and become incorporated into the proviral dna chain , which results in the termination of proviral dna formation.9 the pis operate later in the life cycle of hiv and are designed to stop the protease enzyme from assembling the new hiv material to be released to infect other cells.9 the use of art has become the cornerstone of the clinical intervention that is available to prevent transmission and slow progression of hiv infection in individuals living with hiv / aids . interestingly , efforts have begun for a significant scaling up of the use of art in developing countries , such as those in sub however , questions have been raised about the use of art and how it affects the quality of life ( qol ) of people living with hiv and aids , either negatively or positively . in this review , i examined the effects of art on the qol of people living with hiv and aids , and how art has improved the qol of hiv / aids patients . qol refers to the degree of excellence in a person s life at any given period that contributes to satisfaction and happiness of the person and benefits society . qol is multifaceted , incorporating physical , material , psychological , social , and spiritual well - being.11,12 it is also important to note that qol relates both to adequacy of material circumstances and feeling about these circumstances . it is said to include overall feelings of well - being that are closely related to moral happiness and satisfaction.10 in addition , as health is generally seen as one of the most important determinants of overall qol , it has been suggested that qol may be uniquely affected by specific disease such as hiv / aids.11 reports have shown that hiv / aids patients face various psychological problems , such as stigma , poverty , depression , substance abuse , and cultural beliefs , which can affect their qol not only from the view of physical health but also from that of mental and social health , which can cause problems that affect important activities and interests of the persons.12 in a study that examined coping , social life , and qol among hiv / aids patients , it was observed that income , emotional , social support , and problem- and perception - oriented coping were related to qol.13 another study conducted by swindells et al14 on the relationship between hiv infection and the qol of hiv - infected persons showed that qol was influenced by satisfaction with social support and coping style . the development of art has shifted the perception of hiv / aids from a fatal to a chronic and potentially manageable disease . art is capable of improving survival , reducing the occurrence of hiv - related opportunistic infections , and improving patients qol.15 clinical improvement of hiv - infected patients under art has often been measured by reduction in mortality , opportunistic infection rates , or severe aids - related symptoms.16 however , overall assessments of qol among people living with hiv / aids have also become a major focus of interest as more efficacious and simpler regimen treatments have become available . scientifically and clinically , art has been shown to be highly effective and has the ability to effect significant benefits , which in spite of some negative effects have a positive global impact on the qol and general health of people living with hiv and aids.17 according to mannheimer et al,18 significant improvements in mean qol were seen in hiv patients enrolled at two multicenter antiretroviral clinical trials after 1 and 4 months on new art regimens , and the improvements persisted for 12 months . in a study by campos et al,19 by using a single facet of the whoqol - bref instrument , they were able to assess patients qol for 4 months after initiating art based on the individuals perceptions , values , and preferences , and they found that a high proportion of the patients reported good or very good qol after about 4 months of art ( 66.4% ) and noticed a significant difference when compared to the baseline values before initiating art treatment . overall self - perception of qol has been shown to be a useful screening item for assessing global qol , and a lower proportion of good and very good qol has been observed in hiv / aids patients who are not seeking primary health services , compared to individuals who have sought primary health services.20,21,22 previous studies have suggested that a better perception of qol at baseline is a strong predictor of better qol after initiating art , which may relate to a potential carryover effect during the course of treatment.23,24 also , patients with worse qol at baseline could have higher proportions of anxiety and depression symptoms as well as worse clinical condition for aids , and this could negatively affect qol at follow - up , independent of adherence to art . the impact of psychiatric symptoms on the qol of patients with hiv / aids has been reported.25,26 chan et al27 described a significant reduction in psychiatric symptoms when comparing different treatment groups in a representative sample of hiv - infected patients receiving care in the us , including patients receiving art at baseline , follow - up patients , patients who initiated art at baseline , patients who were on art only at baseline , and patients who were not on art , and showed that art potentially improves qol and contributes to enhanced treatment via better adherence to art . treatment - related factors were identified to be barriers to a good qol , and this is consistent with literature that indicates that maintaining the same art regimen during the follow - up period and shows a better qol than using different art.22,28 adherence is a critical component for therapeutic success in hiv infection , while improved qol has been recognized as an important outcome from treatment of hiv ; however , adherence to art has been shown to be the main determinant of biological outcome measures in hiv , including hiv rna level , cd4 lymphocyte count and genotypic resistance.29,30,31,32 adherence has also been found to predict clinical outcome measures in hiv / aids patients as well as mortality , aids progression , and hospitalization.33,34 the association between qol and adherence has not been well studied , but art adherence is known to contribute to the qol of people living with hiv and aids , and qol is in turn believed to positively influence adherence , as persons with better qol may have a greater ability to adhere to their art regimens ; studies have also shown that adherence and qol share some determinants . both qol and adherence have been shown to be associated with hiv rna levels , hiv disease stage , and symptoms . qol and art adherence share an inverse relationship with hiv rna levels : lower adherence rates predict higher hiv rna levels , and this virological failure has been associated with lower qol scores.30,35,36 according to mannheimer et al,18 participants who reported 100% art adherence achieved significantly higher qol scores at 12 months of follow - up when compared to those with poorer art adherence , and qol improved with art treatment and art adherence.37 in the same study , it was demonstrated that qol improved over time in 1000 hiv - infected persons receiving art and the improvement was sustained over the 12-month period of art treatment , but that the improvement was most significant among participants with the highest adherence levels . in a cross - sectional analysis of their results at 4 , 8 , and 12 months after initiation of art regimens , significant differences were seen at 4 months for the mental component of the qol score and at 12 months for both the mental and physical components and in seven of the qol domains . in each case , those who reported 100% adherence had the greatest gains , those with 80%99% adherence levels had smaller benefits , and those with < 80% adherence had lower qol scores than at baseline . in this same study , male gender was also found to be associated with an improved physical component of qol . however , the authors reported that this was not related to differences in access to art treatment , as all patients received art treatment . education , an indicator of socioeconomic status , has also been shown to influence qol of people living with hiv and aids . reports have shown that patients with higher education reported better qol , possibly due to better knowledge about their treatment and disease , access to health services , or functional status.38,39 in this author s opinion , this suggests that art works in combination with such other factors as education and socioeconomic status to effect the desired improvement in the qol of people living with hiv and aids . in a south african study conducted by wouters et al,40 among 268 patients enrolled in south africa s public sector art program for 12 months , it was reported that the physical and emotional qol of the respondents was high and that the improved qol observed was sustained over the 12-month study period . the study also evaluated the impact of adverse effects of medication on the patients physical and emotional qol , and reported that although adverse effects to art significantly and negatively influenced patients physical and emotional qol , their longitudinal data showed that patients reported significantly fewer adverse effects at follow - up than at baseline . the most cited adverse effects ( dizziness , nausea , and skin problems ) were related to mild toxicity , which does not require the discontinuation of art , and adverse effects decrease over time . improvement in the qol domains of cognitive function , physical health , social activities , pain , sleep , feelings , and emotions following art treatment has been reported.41 it has been demonstrated that patients with higher cd4 counts had better qol , especially with regard to physical health.42,43,44 wu et al45 and ware et al46 also reported similar findings , whereas paton et al47 found that the differences between the disease stages and correlations with cd4 counts extended to nearly all the subscales of physical and mental health . however , some investigators have reported absence of a clear association between scores on psychological domains and the stages of hiv infection , thus signifying the need to combine art with psychological intervention.48,49 studies from europe and north america indicate that hiv - infected women frequently become pregnant , and most hiv - infected individuals have fertility desires that change over time.50,51,52 a study in africa indicated that hiv might modify desires but does not eliminate the desire to have children , and that art use may be associated with increased fertility desires among hiv - infected women , possibly through increased hopes and planning for the future.52 the general consensus is that hiv care and art treatment do encourage hiv - infected women to fall pregnant , because of the belief that it would reduce the risk of vertical transmission of hiv , and to avoid the use of potentially teratogenic drugs , thus ensuring optimal outcomes in these women and their children . a study by myer et al53 showed a significant association between the use of art and increased incidence of pregnancy . the authors noted that within 4 years of follow - up , one - third of women who initiated art experienced a pregnancy , highlighting the fact that art could improve the qol of people living with hiv and aids , including hiv - infected women who wish to have children.53 a series of biological and behavioral factors may influence the association between the use of art and increased incidence of pregnancy . it is possible that the rapid improvements in health and qol that take place with art initiation led to increased sexual activity , especially for those with stable partners.54,55 improving health with art use may contribute to increased fertility desires through psychological mechanisms of increased hopefulness about the future and improved mental health , as well as through increase in sexual activity and new partner acquisition.54 it is also possible that improvements in immunological functioning with art increase female fecundity compared to pre - art levels . a study conducted by kabue et al56 showed that art improves the growth and survival of hiv - infected individuals . to confirm this , the authors designed a retrospective cohort study to assess clinical factors associated with growth in hiv - infected children on art in uganda between july 2003 and march 2006 . height and weight measurements taken pre- and post - art initiation for at least 6 months were age- and gender - standardized according to centers for disease control and prevention criteria , and medical records of 749 children receiving art were analyzed . descriptive and logistic regression analyses were conducted to identify covariates associated with the risk of either stunting or being underweight . longitudinal regression analysis with a mixed model using autoregressive covariance structure was used to compare change in height and weight before and after initiation of art . the mean age of the study population at first visit was reported as 7.5 years , while mean height - for - age , weight - for - age , and weight - for - height percentiles at first visit were 8.6 , 7.7 , and 7.9 , respectively . at last visit , mean height - for - age , weight - for - age , and weight - for - height percentiles were 8.6 , 13.3 , and 13.8 , respectively . a baseline weight - for - age z - score of 1 or more was protective against stunting ( odds ratio [ or ] 0.25 , confidence interval [ ci ] 0.180.35 ) , while a baseline height - for - age z - score of 1 or more was protective against becoming underweight ( or 0.75 , ci 0.630.88 ) . children in who stages ii , iii , and iv at baseline were 1.5 times more likely to become underweight ( or 1.51 , ci 1.072.14 ) . the authors noted that initiation of art resulted in improvement in mean standardized weight - for - age z - score and weight - for - age percentile ( p < 0.001 ) , and also that weight - for - age percentile and z - score improved significantly after initiation of art . they concluded that the studied pediatric population gained weight more rapidly than height after initiation of art . there is both clinical and experimental evidence for direct or indirect negative effects of arv agents . some pis have been observed to reversibly inhibit glucose uptake into muscle and adipose tissue cells at the level of the glucose transport molecule glut4 , and pis may also affect hepatic very - low - density lipoprotein secretion through an inhibition of intracellular apoprotein b degradation.56,57 nrtis , especially thymidine analogs , may also have direct toxicity . for instance , fialuridine , the first agent tested in chronic hepatitis b infection was the precedent for nrti toxicity . although the drug was effective , it led to a high incidence of subacute liver failure with symptomatic lactic acidosis , and the toxic effect was found to be mitochondrial toxicity relating to inhibition of the effect of dna polymerase gamma , the dna polymerase involved in mitochondrial function.58 a single - nucleotide polymorphism in the resistin gene has been shown to be associated with a cluster of metabolic alterations , including insulin resistance , dyslipidemia , and lipoatrophy in patients receiving art.59 the identification of lipodystrophy has had significant implications on the management of hiv , and its recognition as a side effect of art has led to reevaluation of the appropriate time to start art , delay initiation of art , and also to different modifications in art for hiv treatment . several qol studies in hiv lipodystrophy have generally shown that patients enjoy a good qol , including good physical functioning , whereas common complaints that manifested as anxiety and depression were mostly related to the stigma associated with morphological changes.60,61 hiv - positive persons may refuse art or reduce treatment compliance as a result of or in order to avoid the side effects , and as a result , they may experience rebound in viral load or the development of viral resistance , which could consequently lead to disease progression and even to an increased risk of disease transmission , probably by a drug - resistant viral strain . the changes associated with lipodystrophy are associated with cardiovascular disease , and some studies have implicated an increased risk for developing symptomatic cardiovascular disease.62,63 previous studies have suggested that lipodystrophy syndrome in hiv - positive persons on art is characterized by subcutaneous fat wasting , visceral fat accumulation , lipid abnormalities , and insulin resistance or glucose intolerance , and all of these affect the qol of people living with hiv and aids.64 an important observation was made by researchers in the multicenter aids cohort study , who had the privilege of following 50 persons from a period prior to hiv seroconversion through initiation and follow - up of art.66 a fall in total , high - density - lipoprotein cholesterol and low - density - lipoprotein cholesterol concentrations was reported after seroconversion and persisted as immune function declined and initiation of art led to increase in total and low - density - lipoprotein cholesterol . ngondi and colleagues reported that combined art increased the oxidative stress in hiv and aids patients receiving art.67 art may increase oxidative stress levels above levels caused by the hiv itself . in hiv infection , oxidative stress may enhance viral replication by activating nuclear transcription factors , which leads to viral gene expression . in hiv - infected adults , zidovudine was shown to promote oxidative damage to dna.67 it is been noted that art may induce an increase in oxidant generation , decrease in antioxidant protection , and a failure to repair oxidative damage . ngondi et al66 also noticed that increased oxidative stress brought about by hiv as well as the use of art was paralleled by significant decreases in glutathione level , albumin , and vitamin c. it should be noted that abnormally high levels of free radicals as well as a simultaneous reduction in antioxidant defense mechanisms can lead to damage of cellular organelles and enzymes and increased lipid peroxidation . under normal conditions , several cellular antioxidant systems exist to defend against oxidative stress and maintain the redox balance of the cell . however , art may reduce glutathione synthesis , enhance glutathione utilization , or limit intracellular reduction of its reduced form and as a consequence of glutathione deficiency , a number of related functions may be impaired , such as a decrease in reducing capacity , protein biosynthesis , immune function , accumulation of lipid peroxidation products , and detoxification capacity.6870 a reduced detoxification capacity in the liver may lead to accumulation of hepatotoxic metabolites , leading to liver damage and thus reduction in the qol of people living with hiv and aids.68,69 weight loss and wasting may still be observed in hiv patients on art . the evolution of art treatment of hiv / aids as a chronic infection / disease presents challenges for patients and health - care professionals , and measures of qol can provide important information in behavioral and clinical studies of art . the relationships between art and multidimensional construct of the qol of people living with hiv and aids is complex ; however , this review provides a framework for practical and theoretical understanding of the relationships , thus contributing to the understanding of the impact of art on the various aspects of the qol of people living with hiv and aids . focus should be on how to maximize the positive effects of art , especially in resource - poor settings , and particularly in developing countries . there is a need to provide more support to hiv / aids patients living in rural areas and to organize awareness programs that can address the issue of stigma and discrimination , since such awareness programs could contribute to a better qol in hiv / aids patients . clinical assessment of adverse reactions during the course of art treatment and careful monitoring after any art switch could contribute to a better qol , improve the patient doctor relationship , and potentially maintain adherence with fewer undesired side effects .

a 7-year - old boy was admitted with a history of diminution of vision in both the eyes following a cracker injury 7 months ago . the acute symptoms had subsided , but there was constant irritation with poor vision . on examination , there were multiple intracorneal foreign bodies , some reaching up to descemet 's with irregularly thinned cornea and leucomatous corneal opacity , in both the eyes [ fig . 1 ] . though anterior segment optical coherence tomography ( oct ) would have been an ideal tool to identify the depth of involvement precisely , the same could not be performed due to nonavailability . best corrected visual acuity ( bcva ) was counting fingers close to face in both the eyes . the peripheral iris was normal , - lens was clear , intraocular pressure ( iop ) was 18 mmhg and fundus could not be visualized although b - scan was normal . the child was posted for anterior lamellar keratoplasty ( lk ) of the right eye under general anesthesia using a donor cornea from a 62-year - old subject who died of cardiac arrest . the reported death to enucleation time ( det ) was 10 h. although this is more than the accepted 6-h limit , but due to the limitation of availability of donor eyes in our country , we often accept the tissues even after the scheduled det of 6 h. these eyes are expected to have suboptimal endothelium for which these are either used for therapeutic / tectonic keratoplasty . it was preserved in the mccarey - kaufman medium for 14 h prior to transplantation . clinical photographs with multiple corneal opacities no specific preoperative medication like lowering of iop was necessary as required in penetrating keratoplasty . accordingly , an 8.5-mm diameter and a 0.3-mm ( approximately ) depth partial thickness anterior lamellar dissection was carried out [ fig . 2 ] . the recipient dissection was performed manually , with the help of a crescent blade , due to irregular thinning of the cornea . however , care was taken to remove maximum number of foreign bodies to make the visual axis and pupillary area clear . photograph showing partial thickness anterior lamellar dissection moria automated lamellar therapeutic keratoplasty ( altk ) artificial anterior chamber ( moria / microtek inc . doylestown , pa , usa ) was used to mount the donor corneoscleral tissue button . a plate with a central opening and a sliding platform was fixed . the microkeratome head ( 350 ) was used to obtain an anterior partial thickness corneal button of 8.75 mm diameter to be used for lk [ fig . 3 ] . the anterior partial thickness corneal button removed by microkeratome from the donor cornea after giving a thorough wash to the recipient bed to remove the tissue debris , blood clot if any , or any other foreign bodies , the field was dried with a cellulose acetate sponge . freshly prepared fibrin glue solution ( reliseal , relinace life sciences , india ) was spread uniformly over the recipient bed and the donor button was placed over the recipient bed . uniform and firm pressure was applied from the center to the periphery and more toward the edge [ fig . 4 ] . the subconjunctival injection of gentamycin ( 20 mg in 0.5 ml ) and dexamethasone ( 2 mg in 0.5 ml ) was given and a firm pad and bandage was applied to complete the surgery . partial thickness corneal button glued to the recipient 's cornea the bandage was opened after 24 h and the eye received 1% prednisolone acetate eye drops every 2 h in the first week , every 4 h for the next 2 weeks , four times a day up to 12 weeks , twice a day up to 6 months , and then it was stopped . similarly , 0.3% gatifloxacin eye drops were instilled four times a day for 2 weeks . the patient was followed up everyday for 3 days , every week for 2 weeks , every month for 3 months , and every 3 months thereafter . 6 months post operatively , the patient was symptom free with a graft clarity of 3 + and bcva of 20/60 with -1 d sphere and -1d cylinder 120. on slit - lamp examination , there was a mild interface haze but no vascularization at the graft host junction , despite there being a few preexisting superficial vessels in the recipient eye . it is well tolerated by the patients , nontoxic to the tissue wherever it is applied and has some antiinfectious activity . it allows a smooth seal along the entire length of the wound edge and because of the shear adhesive strength , it maintains the tissue fixed to the surface . in corneal surgery it has been used for varied applications such as amniotic membrane transplantation for ocular surface reconstruction , limbal stem cell transplantation , epikeratophakia , and as an aid to stabilize temporarily sutured keratoprosthesis . its use was also advocated for corneal graft.[811 ] rosenthal et al . used a platelet / fibrinogen / thrombin mixture to fixate the lamellar corneal graft in experimental animals . the average incisional bursting pressure was 185 mmhg ( range 90300 mmhg ) for testing stability . narendran et al . carried out deep lk using fibrin glue supported with overlay sutures . a laboratory study was carried out by bahar et al . in 2007 in top hat keratoplasty and they found fibrin glue mechanically more stable than suturing . in the present communication , we evaluated the efficacy of fibrin glue in graft fixation in anterior lk . we found the technique as a safe and an effective one in terms of less surgical time and negligible postoperative reaction . furthermore , postoperative graft vascularization , a leading cause of graft failure , was also not seen . thus , we recommend that suturing should no more be an automatic choice during lk . however , more number of cases with longer follow - ups in years should be performed to find out the postoperative problems and wound stability . it would be more appropriate if the technique can be performed and compared by more number of experienced corneal surgeons ( preferably on lk ) to highlight its efficacy .

stroke risk in patients with symptomatic internal carotid artery ( ica ) stenosis greater than 70% can be reduced effectively by carotid endarterectomy . in most cases , carotid ultrasound is performed in association with tia or stroke and , in case of significant stenosis , an operative treatment or pharmacotherapy is initiated as a secondary prevention . additionally , asymptomatic patients with carotid stenosis could benefit from primary preventive operative treatment with carotid endarterectomy in presence of a hemodynamically significant 6099% ica stenosis [ 13 ] . a number of patients needed to be treated with this method are approximately 20 , to prevent one stroke in 5 years . even in mild carotid stenosis ( < 50% ) silent embolic infarcts predict also future ipsilateral stroke in at least moderate asymptomatic carotid stenosis , and elderly with moderate carotid stenosis had a significant ipsilateral cerebral perfusion delay on mri . it is still unclear to what extent the mild carotid stenosis is responsible for , or correlates with silent vascular brain changes . calculating carotid stenosis to less than 50% measurement of peak systolic velocities ( psvs ) in ica has been used worldwide , and maximal psv , defined between 1.251.4 m / s , was used delimitate stenosis to maximum 50% , sometimes with mandatory plaque or intimal thickening visible . when estimating cerebral blood supply and carotid stenosis with ultrasound in the elderly , one must be aware , that age and dbp - level decrease psv , while sbp and pulse pressure increase psv in elderly [ 8 , 9 ] . in elderly with cerebral ischemia , larger diameters of common carotid artery and lower psv have been observed , probably due to increased intracerebral circulatory resistance [ 10 , 11 ] . at the same time , the risk of stroke was highest in subjects with increased psv in middle cerebral artery . changes in blood pressure in the very elderly could also affect psv , mainly due to a frequently observed blood pressure decline after age 75 , or decreased cardiac dynamic parameters . the aim of our study was to estimate the presence of silent changes in cerebral blood flow ( cbf ) in a populations sample of stroke - free elderly men without carotid stenosis or less than 50% and to examine if there was a dose - response relationship between psv in ica and a grade of cbf decline . it includes all men born in the even months of 1914 in the city of malm , sweden . a total of 809 men were invited to participate in the study , and 703 men took part in the first health examination . when they were 68 years old , 465 men in the cohort and an additional 95 new residents were invited to attend a new examination . five hundred of them agreed to participate in this first followup ( figure 1 ) . the most recent / second followup of the cohort started when the subjects reached 81 years of age , and 281 men were found to be still alive . of these , 185 agreed to take part ( 66% ) in a new investigation , including both physical and psychological examinations as well as carotid ultrasound bilaterally . one year later , all 185 subjects were invited to a measurement of cerebral blood flow with spect , but only 129 of them agreed to participate . six subjects had suffered from a stroke previously , according to the local stroke register , and were excluded from the study . vascular risk factors , including hypertension , levels of blood glucose , cholesterol , and triglycerides during fasting conditions as well as body mass index ( bmi ) were measured at age 68 . blood pressure was measured sphygmomanometrically with korotkoff method in the upper right arm in the supine position after 15 min of rest ( at age 68 ) and in the sitting position ( at age 81 ) , using a calibrated mercury manometer and rubber cuffs ( 12 35 cm for normal and 15 cm for obese subjects ) . peripheral circulation in the lower extremities was estimated using the ankle - brachial pressure index ( abi ) at age 68 and 81 ( see below ) . at the recent followup at age 81 , the medical examination was repeated , and 185 men answered a questionnaire focusing on lifestyle and health markers . possible dementia was classified according to the dsm - iv criteria and , according to them , one subject was diagnosed as being demented . the mini - mental state examination ( mmse ) was performed on 171 men , giving a mean value of 28.23 1.85 ( sd ) ; range 1830 . hypertension was defined as systolic and diastolic brachial bp 160 mmhg or 90 mmhg , respectively , or medication for hypertension . these hypertension criteria have been used previously and were valid until the world health organization drew up new ones in 1999 . all the subjects had been monitored and treated during their lifetime according to these hypertension criteria , and therefore they were used for the statistical analysis . the carotid arteries were examined at age 81 , using a computed sonography system ( acuson xp 10 , acuson , mountain view , ca , usa ) with a 7 mhz b - mode real - time linear scanner , including a 5 mhz pulsed and color - coded doppler . the color - coded doppler was used to identify areas with high flow velocities in the internal carotid artery ( ica ) , and the maximum flow velocity ( m / s ) was measured with the pulsed doppler . the angle between flow direction and doppler signal was carefully corrected and always kept below 65. the degree of stenosis in the ica was determined from the peak systolic velocity to the equation : y = 0.54 e , where y is the peak systolic velocity in the ica in m / s , and x is the degree of stenosis expressed as the diameter reduction in percentage . diameter reduction in percent = [ b a ] 100 , where a is the smallest diameter in the stenotic zone , and b is the diameter of normal common carotid artery proximal to the stenosis . each subject received an intravenous injection of 800 mbq 99mtc - hmpao ( ceretec ; amersham inc . the acquisition was performed under resting conditions on a triple - headed gamma camera system ( siemens multispect 3 , siemens , chicago , il , usa ) with fan beam low - energy collimators and in 360 rotation ( 64 views , 20 s / view , in a 128 128 matrix , and a zoom factor of 1.23 ) . the energy window used had a 15% window centered over the 140 kev peak . image processing included reconstruction of 10 transaxial 1 cm - thick slices , from 1 cm below the orbitomeatal line and upwards . regions of interest were delineated in each slice and defined as right or left : frontal , temporal medial , temporal lateral , parietal superior , parietal inferior , basal ganglia , and subcortical as in figure 2 . cbf was expressed as a regional count density percentage of the mean cerebellar count density . ankle blood pressure was estimated , at the ages of 68 and 81 years , by placing a cuff around the ankle and using a doppler signal on the tibial posterior artery or dorsal foot artery to detect peripheral blood flow . both ankle pressure and the reference pressure in the arm were calculated using the strain gauge system . the occluding cuff was placed on the arm , and finger blood flow was continuously recorded by mercury - in - silastic strain gauge pulse sensor placed on the proximal phalanx of the first digit . wheatstone bridge with amplifier was used to record the resistance of the strain gauges , and a pressure transducer ( siemens - elema emt 746 with amplifier emt 311 ) was used to record cuff pressures . arithmetic average of a two recordings was used . the ankle - brachial pressure index ( abi ) was calculated for each leg by dividing the ankle sbp by the highest individual upper arm sbp value . partial correlation has been calculated between variables when controlling for the grade of carotis stenosis . the mann - whitney u test for independent samples was used to analyze differences . the study was approved by the local ethics committee at lund university ( lu 111 - 82 ) . the data of the cohort men born in 1914 are presented in table 1 . carotid stenosis 50% was more frequent in the right ica than on the left side ( 11.6% versus 5.7% ) , and mean psv was also higher in the left than in right ica . however , the vast majority of this population sample was composed of subjects with carotid stenosis less than 50% , and this subgroup has been chosen for further analyses . in subjects with carotid stenosis < 50% on the right side , 22 were nonstenotic out of the 116 , while on the left side , 21 were nonstenotic out of the 110 subjects . blood pressure data , between the first followup at age 68 and at the end of the study at age 81 , showed an overall sbp decline with mean 7.2 mmhg , and dbp decline with 9.6 mmhg . ankle - brachial index ( abi ) decreased with mean index .10 in the right and .09 in the left leg during the followup . in a whole cohort the presence of long - term / early hypertension has been chosen as a grouping factor , and the cohort has been divided into hypertensive and normotensive subgroup at age 68 . in subjects who were found normotensive at 68 , both left and right carotid psv at age 81 correlated negatively with cbf in several brain areas . in 110 subjects with left - sided carotid stenosis < 50% , 59 were normotensives , and negative correlation has been observed between psv in left ica and cbf in frontal , mesial temporal , inferior and superior parietal and subcortical areas ( table 2 , 1st column ) . in the 57 normotensives with right - sided carotid stenosis < 50% , psv in left ica correlated negatively with cbf in frontal , mesial temporal , inferior and superior parietal , and subcortical areas as well as in basal ganglia ( table 2 , 2nd column ) . in both carotids , highest correlation coefficients were observed with cbf in mesial temporal areas , bilaterally ( figure 3 ) . in those who were hypertensive at age 68 , no significant correlations have been observed between psvs and regional cbf , either on the left side ( 56 subjects with left ica stenosis < 50% ) or the right side ( 59 subjects with right ica stenosis < 50% ) ( table 2 , columns 3 - 4 ) . to analyse if the relationship between psv and cbf is independent in carotid stenosis , a partial correlation has been calculated between psv and cbf controlling for carotis stenosis in % . the data showed a nonaltered results for right ica and moderately diminished scope for left ica ( significant for 3 instead of 8 brain areas ) ( table 3 ) . similarly , as in nonadjusted analysis , no significant correlations have been observed between psv and cbf in hypertensives at age 68 after controlling for carotid stenosis . to explain the different trends in psv - cbf relationship by the presence of an early hypertension , vascular characteristics in the normotensive and hypertensive subgroups have been analysed . psvs did not differ significantly between normotensive and hypertensive subjects ( left ica : 0.71 0.18 versus 0.69 0.16 , p = 0.455 ; right ica : 0.67 0.19 versus 0.67 0.16 , p = 0.978 ) . when comparing psvs in all 160 survivors , who were examined by ultrasound but before inclusion in the cbf , normotensive subjects presented significantly higher psvs in left ica ( 0.72 0.18 versus 0.67 0.16 , p = 0.035 ) and slightly larger , but nonsignificant , difference in right ica ( 0.69 0.19 versus 0.65 0.16 , p = 0.213 ) . in normotensive subjects at age 68 , left - sided psv correlated positively with sbp at age 81 and with time difference in sbp between ages 81 and 68 , showing highest left - sided psv values in those subjects who developed largest sbp - increase during 13-year followup ( table 4 ) . this relationship was not observed in hypertensive subgroup at age 68 . pulse pressure levels at age 81 correlated strongly positively with left - sided psvs on both sides in normotensive , but not in hypertensive subgroup . psv correlated also negatively with abi in both legs at age 81 , both in normotensive ( left ica ) and hypertensive at age 68 ( right ica ) . also a lower abi , estimated at age 68 , predicted higher psv values in both subgroups . the time trend of abi , expressed as a difference between individual values measured at 81 and 68 , correlated negatively with psv , that is the larger decline in abi over observation time , the higher were the psvs ( table 4 ) . subjects who were normotensive already at age 68 , differed from hypertensive ones , by lower pulse pressure at 68 ( 52.1 versus 64.4 mmhg ; p < 0.001 ) , and at 81 ( 58.1 versus 63.8 mmhg ; p = 0.015 ) . normotensives had also more stable blood pressure during 13-year followup , compared to a marked blood pressure decline in hypertensives ( sbp difference age 8168 . normotensives : 1.5 17.5 mmhg versus hyper - tensives : 14.9 21.2 mmhg ; p < 0.001 ) ( dbp difference age 8168 . normotensives : 4.4 8.6 versus hypertensives : 14.4 8.5 mmhg ; p < 0.001 ) . abi at age 68 did not differ between normo- and hyper - tensives , but at age 81 , abi was higher in normotensives in the left leg ( 1.04 0.22 versus 0.95 .17 ; p = 0.005 ) , and abi time decline from age 68 to 81 was lower than in hypertensives ( left abi difference , age 8168 : 0.05 0.13 versus 0.13 0.16 ; p = 0.007 ) . values of cbf did not differ between normotensive and hypertensive subjects diagnosed at age 68 ( table 5 ) . in our study cohort , comprising 123 stroke - free men who reached the age of 82 years and performed cbf measurement , the frequency of a moderate- or high - grade carotid stenosis was very low . survival analysis performed previously in this cohort showed an association between early - onset carotid stenosis at age 68 with a higher mortality from ischaemic heart disease during following ten years , especially when combined with low abi . then , due to the selective mortality , the population - based sample of octogenarians studied here has low frequency of high - grade carotid stenosis . some of the disabled elderly chose not to join the last reexamination or were not capable to be examined due to disabilities or hospitalization . a dropout analysis has been published earlier , and the background data did not differ between these groups concerning : sbp , dbp , presence of hypertension , ankle - arm indexes , b - glucose , triglycerides and cholesterol levels , prevalence of accumulated vascular events , smoking history at age of 68 years ; prevalence of stroke up to age of 82 years , or difference in sbp or dbp between ages 68 and 82 years . however , study subjects were invited in a randomized way and are fairly representative of the men of their age in the community . our findings ignore also a possible confounding effect of antihypertensive medication , when comparing hyper- and normo - tensives . it is unclear if increased psv in ica should be regarded as a risk factor for silent brain damage , or if it is a reverse marker of increased cerebrovascular resistance . generally , low , not high ica flow velocities are treated as indicators of higher cerebral vascular resistance . in acute as well as chronic stable patients with ischemic stroke , there were significantly lower psvs and higher resistance index compared to the nonstroke patients , independently of extracranial carotid atherosclerosis [ 9 , 10 ] . on the other side , in subjects with psv < 1.25 m / s and the presence of a sonographic atherosclerotic plaque < 50% , a significantly higher frequency of both 1- , 2- and 3-vessel coronary artery disease has been observed . in the common carotid , internal carotid and the vertebral arteries flow velocities this age - related enlargement in cross - sectional area results from stretching of elastic fibers in the vessel wall or an altered baroreceptor reflex . parallely , an age - related decline of intravascular flow volume was observed in the ica . due to a pronounced decrease in end - diastolic flow velocity , the resistance index decreased during ageing . while ica 's psvs are generally decreasing with age , the role of hypertension is contradictory . in a majority of studies , an enhancing effect of hypertension was observed . except inverse correlation with age , peak common carotid artery velocity in the elderly people was also inversely associated with diastolic blood pressure and directly associated with pulse pressure , while ica psvs decrease with advancing age and increase with increasing pulse pressure . as the effects of systolic and diastolic pressure seems to be opposing , pulse pressure , which summarize their combined effect , is related to psv increase by 0.21 cm / sec / mm . in our study , psv correlated also with abi , and was higher in subjects who had mostly aggravating atherosclerotic process in peripheral arteries , expressed by decreasing abi over the last 13 years . during early examination of the same cohort , an abi < 0.9 was associated with a 2.4 times higher total mortality and a 2.0 times higher cardiac event rate . knowing that high pulse - pressure is a risk factor for end - organ damage , that is cerebrovascular disease , in our cohort , high psv in subjects with low cbf can reflect deleterious effect of high pulse pressure and/or express vascular stiffness . knowing that psvs generally decrease with age , in some very elderly , an effect of arterial stiffness expressed by high pulse pressure may reverse this trend and lead to higher psvs , or just erase an age - related psv decline by reflecting higher atherosclerotic load in intracranial vessels and in cbf . as speculated , the autoregulatory mechanisms of cbf could blunt velocity changes in the carotid artery to maintain a constant pressure gradient between the cerebral arteries and the brain . in case of high pulse pressure , cerebral vasoconstriction may be down - regulated to maintain flow during the longer lower - pressure diastolic phase of the cardiac cycle . the net effect of this decrease in downstream resistance would be to enhance flow and increase velocities in the setting of an elevated pulse pressure . the effect of pulse pressure must overcome higher cerebral vascular resistance , which is expressed by low , not high , ica flow velocities . it has also been reported that , in acute as well as chronic stable patients with ischemic stroke , there were significantly lower flow velocities and higher resistance index compared to the nonstroke patients , independently of extracranial carotid atherosclerosis [ 9 , 10 ] . cardiac function affect psv by a direct correlation between psv in the left cca and ica and fraction shortening of heart muscle , and between psv in the left cca and peak velocity on left ventricular outflow tract . assuming that heart failure is more frequent in subjects with cerebrovascular disease , the psvs should have an opposite relation to cbf than observed in this study . probably , in relatively healthy octogenarians of this cohort , this mechanism was attenuated by presence of heart failure in subjects who were still normotensive at age 68 and who developed a mild hypertension at age 81 , while absent in those who had an early hypertension , reached an inflection point in their seventies and expressed lower blood pressure levels at the time of ultrasound examination . in our cohort , psv values were higher on the left side in normotensives at age 68 , who in their majority developed blood pressure increase during followup , compared to hypertensives whose blood pressure generally declined until age 81 . highest psv values have also been observed in those subjects who had high sbp at age 81 , and most increasing sbp values during last 13 years . these findings were limited to subjects who were normotensive at the beginning of the observation at age 68 . this subgroup had also equal values of decreasing and increasing sbp during followup , while hypertensive at 68 registered predominant blood pressure decline until age 81 , especially concerning dbp . in hypertensives at age 68 , contrary to normotensives , psvs were even negatively correlated with dbp values , as it was observed in other studies . decreasing blood pressure and lower psvs in formerly hypertensives could reflect incipient heart failure or dysautonomia . on the whole , these results suggest that our population of octogenarian men consists of two different groups concerning blood pressure trend , resulting in a different cause - effect relationship between ica 's psvs and regional cbf . firstly , elderly with late - onset hypertension ( after 68 years ) with increasing sbp values over time had slightly higher psv , mainly on the left side and a clear relationship between high psv in carotids , the grade of systolic blood pressure increase and cbf defects of arteriosclerotic origin probably due to dynamic effect of high pulse pressure , which simultaneously affects cerebral circulation . secondly , subjects with early - onset hypertension , with blood pressure decline over time , either due to dysautonomia or heart failure , where lower blood pressure in the eighth decade express higher vascular lifetime - load , and where higher peripheral resistance in cerebral vessels and lower cardiac output could blur the pulse pressure related psv increase and its effect on cbf . to our knowledge , this is a first study showing a correlation between high peak systolic velocities in the internal carotid artery and a widespread cerebral blood flow defects in stroke - free very elderly men with carotid stenosis less than 50% . this observation was limited to those who were normotensive and untreated until their seventies and in majority developed late - onset hypertension with subsequent blood pressure and pulse pressure growth . elderly men with velocities between 0.71.3 m / s should be offered an intensified pharmacotherapy and lifestyle modification .

the incidence of mesenteric and omental cysts is 1 in 20,000 among children , and even lower among infants . of these two types of cystic mass , only 2.2% are omental cysts . reported cases in the literature usually display symptoms of abdominal distension , abdominal pain , painless mass , or possible ascites . mesenteric and omental cysts are classified into four main groups : embryological , traumatic , neoplastic , and infective or degenerative . a previously healthy five - and - a - half - year - old boy was evaluated at the hospital because of abdominal distension that was noticed by his family . septated ascites was noted on ultrasonography , and further investigation was done with computerized tomography ( ct ) , which revealed a mass . tuberculosis peritonitis was the suggested diagnosis because of the radiological findings and a history of tuberculosis exposure from a close family member during infancy . a tuberculin purified protein derivative test , paracentesis for bacteriological tests , and cytology for malignancy were all negative . antituberculosis treatment was given for nine months because of the family history of tuberculosis , despite fluid from the ascites being negative for tuberculosis , but he gained no benefit from this treatment . for the following two years , he did not receive any treatment , as the family did not seek medical help . the patient was admitted to our hospital for painless distension of the abdomen for three years , but without any complaints . his weight - for - age and height - for - age were in the normal percentiles . ultrasonography revealed massive intraperitoneal fluid with septa extending from the subdiaphragmatic region to the pelvic floor . diagnostic paracentesis was performed and 80 ml of serous fluid was removed , which proved to be transudate , with lactate dehydrogenase of 126 u / l , protein of 4.27 g / dl , triglycerides of 15 mg / dl , specific gravity of 1008 , ph of 7.5 , a fluid - protein / serum - protein ratio of 0.6 , and equal serum and fluid glucose levels ( 99 mg / dl ) . magnetic resonance imaging ( mri ) demonstrated significant fluid collection in the peritoneal cavity , displacing bowel loops centrally with the absence of bowel loop separation ( figure 1a - c ) . no solid components were seen , and no enhancement was observed after administration of contrast . diffusion - weighted images showed different diffusion properties of the fluid between the left and right parts of the cyst . the cystic contents of the left portion showed restricted diffusion , with dense fluid content ( figure 1d ) . a 25 20 cm cyst originating from the omentum was dissected from the surrounding structures . the mass was bilobulated , with a difference in color of the two parts ( figure 2a ) . the resection specimen was a multilocular cystic mass weighing 1,000 g , filled with serohemorrhagic fluid , and covered with a thin transparent wall . the microscopic features were consistent with lymphangioma ; the cyst arising from the omentum was lined with an endothelial layer , and some areas of lymphocyte aggregates were detected within the stroma ( figure 2b ) . findings suggestive for tuberculosis , such as granuloma formation , were not present , and no acid - fast bacilli were detected by ziehl the incidence of mesenteric and omental cysts is 1 in 20,000 among children and lower in infants . of these two types of cystic mass , different etiologies have been discussed regarding the precise causes of these cysts , including benign proliferation of mesenteric lymphatics , failed fusion of the mesenteric leaves , and deficiency of the normal lymphaticovenous shunts ( 1 - 3 ) . lymphangioma is the most common cause of these cysts , which are generally restricted to the lesser or greater omentum ( 2 , 3 ) . mesenteric and omental cysts are classified into four main groups : embryological , traumatic , neoplastic , and infective or degenerative . the overwhelming majority of these cysts are mesothelial , lined by mesothelium as well as endothelial cells , and most contain serous fluid , which leads to the misdiagnosis of ascites ( 2 , 4 , 5 ) . patients admitted to the hospital may be classified into two main groups : those with acute clinical symptoms and those with non - acute clinical symptoms ( 4 ) . although symptoms correlate to the location and size of the cyst , non - acute clinical symptoms include painless abdominal mass , abdominal pain , abdominal distention , and possible ascites ( 1 , 3 , 5 ) . diagnosis of a cyst should be considered even if the findings are non - specific and the patient exhibits symptoms over a long period of time ( 6 ) . the presence of complicating factors , including hemorrhage , torsion , infection , rupture , or pressure to other structures , is relevant with acute presentations that require urgent surgery ( 4 ) . sonographic findings frequently feature multiloculated , fluid - filled , and predominantly cystic lesions ( 2 , 3 , 7 ) . mri and ct may provide additional information for determining the detailed definition , exact extension , and characterization of the lesions ( 1 , 3 , 8) . the signal pattern of lymphangiomas on mri resembles that of fluid : low signal intensity on t1-weighted images and high signal intensity on t2-weighted images . the presence of proteinaceous , blood , or fat components within the lesion may alter the ct attenuation and mri signal patterns . there are no specific findings for lymphangioma on diffusion - weighted mri . in our case , restricted diffusion was seen in the left part of the cyst , revealing the dense fluid content , but there was no signal difference in the different parts of the lesion on the t1 sequence . the lack of fluid in the dependent recesses of the peritoneum , clustered bowel loops , and the absence of fluid separation between bowel loops are essential signs for differentiating between ascites and cystic lesions . choledochal cysts , splenic cysts , multicystic dysplastic kidneys , intestinal duplication cysts , and ovarian cysts are all cystic lesions that can be included in the differential diagnosis of omental cysts ( 3 , 9 ) . malignant transformation of cystic lesions is also rare ( 1 , 3 , 9 ) . laparoscopic management has the advantages of lower cost and decreased morbidity compared to open surgery ( 1 , 3 ) . omental cysts are rare pathologies during childhood and can mimic ascites . clinicians should consider this diagnosis for patients with abdominal distension and a cystic mass in the abdomen .

a previously healthy 62-year - old male presented with a 9-day history of severe headache and vomiting . on admission , his neurologic examination was normal , except for a stiff neck . the patient was judged clinically to be hunt and hess grade i. the brain computed tomography ( ct ) revealed subarachnoid hemorrhage ( sah ) prominently occupying the right sylvian fissure and suprasellar cisterns . the ct and angio - ct findings indicated that the right mca aneurysm was probably responsible for the sah . angio - ct of pre- and postoperation : ( a ) cerebral angio - ct revealed 2 aneurysms arising from the bmca bifurcations , and ( b ) postoperative angio - ct confirmed good occlusion of the bilateral aneurysms . a standard right supraorbital keyhole approach with small posterior extension for frontobasal lateral burr hole is used for bmca aneurysm clipping . the patient is positioned supine , and the head fixed in a mayfield frame is slightly titled backward and rotated 20 away from the right side . an approximately 5-cm skin incision was made in the upper half of right eyebrow starting from the midpupillary line to behind the frontal process of the zygomatic bone . the temporalis muscle was cut approximately 1.5 cm in the lateral part of the incision , and exposed for a frontobasal lateral burr hole posterior to the temporal line . the minicraniotomy ( approximately 3 3 cm ) is performed in the frontal bone . the dura is then opened in a curved fashion with its base toward the roof of orbital providing a 2.5 cm opening . with the use of a narrow brain spatula , the right frontal lobe was gently retracted . to avoid frontal lobe contusion spatula creeping technique was adopted until the optic nerve , chiasmatic , lamina terminalis , and internal carotid artery ( ica ) cistern are encountered , at which point the cisterns are opened to achieve brain relaxation and frontal lobe retraction . the 30 endoscope was then placed along the bottom of frontal lobe , and fixed by a holding device allowing bimanual manipulation during the surgical procedure . subsequently , arachnoid attachments between the frontal lobe and olfactory nerve are dissected to avoid postoperative anodmia . after the right ica and sylvian cisterns are opened , the right ica bifurcation was identified , and then dissection is continued by following m1 until the right aneurysm was encountered . after careful dissection of aneurysm neck free of adhesions to the surrounding vascular and neural structures , the right aneurysm was clipped ( fig . afterward , the endoscopy was adjusted to the left side and anterior communicating artery complex and the left optic nerve was identified , and the dissection is continued to contralateral ica bifurcation by following the contralateral a1 segment . the left ica and sylvian cisterns are then opened , and finally the contralateral m1 segment is identified . dissecting along the m1 from medial to lateral direction , the left aneurysm and the 2 m2 branches were visualized . the aneurysm was clipped without temporal clipping of the m1 ( fig . the procedure of clipping bmca aneurysms : ( a ) intraoperative picture of the right sylvian cisterns dissection , ( b ) intraoperative picture of clipping the right aneurysm , ( c , d ) contralateral progression of the dissection the left ica bifurcation and sylvian fissure , ( e ) intraoperative picture of left aneurysm , and ( f ) intraoperative picture of clipping the right aneurysm . a. , internal carotid ; m1 , 2 , middle cerebral artery segment m1 , 2 ; optic . the patient was admitted to intensive care unit for the first 24 hours after operation . calcium channel biockers ( nimodipine ) had been given at 4 ml per hour via venous pump , and 3h therapy ( hypertension , hypervolemia , and hemodilution ) was adopted to elevate systolic blood pressure to150 to 160 mm hg , to maintain intravenous infusion volume 200 ml per hour , and to keep hematocrit 35% . the patients underwent brain ct examination after anesthetic recovery , and postoperative angio - ct 5 days after operation . a previously healthy 62-year - old male presented with a 9-day history of severe headache and vomiting . on admission , his neurologic examination was normal , except for a stiff neck . the patient was judged clinically to be hunt and hess grade i. the brain computed tomography ( ct ) revealed subarachnoid hemorrhage ( sah ) prominently occupying the right sylvian fissure and suprasellar cisterns . the ct and angio - ct findings indicated that the right mca aneurysm was probably responsible for the sah . angio - ct of pre- and postoperation : ( a ) cerebral angio - ct revealed 2 aneurysms arising from the bmca bifurcations , and ( b ) postoperative angio - ct confirmed good occlusion of the bilateral aneurysms . a standard right supraorbital keyhole approach with small posterior extension for frontobasal lateral burr hole is used for bmca aneurysm clipping . the patient is positioned supine , and the head fixed in a mayfield frame is slightly titled backward and rotated 20 away from the right side . an approximately 5-cm skin incision was made in the upper half of right eyebrow starting from the midpupillary line to behind the frontal process of the zygomatic bone . the temporalis muscle was cut approximately 1.5 cm in the lateral part of the incision , and exposed for a frontobasal lateral burr hole posterior to the temporal line . the minicraniotomy ( approximately 3 3 cm ) is performed in the frontal bone . the dura is then opened in a curved fashion with its base toward the roof of orbital providing a 2.5 cm opening . with the use of a narrow brain spatula , the right frontal lobe was gently retracted . to avoid frontal lobe contusion spatula creeping technique was adopted until the optic nerve , chiasmatic , lamina terminalis , and internal carotid artery ( ica ) cistern are encountered , at which point the cisterns are opened to achieve brain relaxation and frontal lobe retraction . the 30 endoscope was then placed along the bottom of frontal lobe , and fixed by a holding device allowing bimanual manipulation during the surgical procedure . subsequently , arachnoid attachments between the frontal lobe and olfactory nerve are dissected to avoid postoperative anodmia . after the right ica and sylvian cisterns are opened , the right ica bifurcation was identified , and then dissection is continued by following m1 until the right aneurysm was encountered . after careful dissection of aneurysm neck free of adhesions to the surrounding vascular and neural structures , the right aneurysm was clipped ( fig . afterward , the endoscopy was adjusted to the left side and anterior communicating artery complex and the left optic nerve was identified , and the dissection is continued to contralateral ica bifurcation by following the contralateral a1 segment . the left ica and sylvian cisterns are then opened , and finally the contralateral m1 segment is identified . dissecting along the m1 from medial to lateral direction , the left aneurysm and the 2 m2 branches were visualized . the aneurysm was clipped without temporal clipping of the m1 ( fig . the procedure of clipping bmca aneurysms : ( a ) intraoperative picture of the right sylvian cisterns dissection , ( b ) intraoperative picture of clipping the right aneurysm , ( c , d ) contralateral progression of the dissection the left ica bifurcation and sylvian fissure , ( e ) intraoperative picture of left aneurysm , and ( f ) intraoperative picture of clipping the right aneurysm . a1 , anterior cerebral artery ; car . a. , internal carotid ; m1 , 2 , middle cerebral artery segment m1 , 2 ; optic . n. , optic nerve . the patient was admitted to intensive care unit for the first 24 hours after operation . calcium channel biockers ( nimodipine ) had been given at 4 ml per hour via venous pump , and 3h therapy ( hypertension , hypervolemia , and hemodilution ) was adopted to elevate systolic blood pressure to150 to 160 mm hg , to maintain intravenous infusion volume 200 ml per hour , and to keep hematocrit 35% . the patients underwent brain ct examination after anesthetic recovery , and postoperative angio - ct 5 days after operation . surgical procedure was uneventful , and the patient had an excellent postoperative course without neurologic impairment and complication . approximately 7% to 34% of the patients harbor multiple intracranial aneurysms , and the aneurysms are bilateral in 20% to 40% of these patients . the incidence of bmca aneurysms aneurysms has been reported approximately 1% of all intracranial aneurysms . middle cerebral artery aneurysms are generally not suitable for endovascular treatment , due to their often complex configuration and/or broad neck . therefore , surgical treatment is still the first choice in many patients with mca aneurysms . the contralateral approach can be considered as a useful procedure to clip bmca aneurysms ; its use is not widely spread because of technical difficulty , the long dissecting distance , limiting view , and impaired the brain tissue . oshiro et al previously described the contralateral exposure for the most common sites of anterior circulation aneurysms . several authors have reported clipping of bilateral aneurysms via unilateral craniotomy successfully in case series and clinical reports . surgical management of bmca aneurysms is still controversial and considered a challenge procedure , although clipping all aneurysms in a single - stage operation is more preferred . the unilateral approach presents some important advantages comparing 1- or 2-stage operations : avoiding a second craniotomy , shorter operation time , shorter hospitalization , reducing the cost of treatment , and less cosmetic defect due to single craniotomy . in addition , the most important advantage of 1-stage operation is elimination of the risk of rebleeding from unrupture aneurysms , due to 3h treatment for vasospasm may carry a potential risk of rupturing an untreated aneurysm in the postoperative period . the recent keyhole approaches have been made possible by technological advances such as by three - dimensional ct angiography guidance , by the use of endoscopes , and by the manufacturing of dedicated microsurgical instruments . the supraorbital keyhole approach is a minimally invasive approach , and both the ipsilateral and contralateral mca aneurysms can be approached from proximal to distal . the side of approach was preferentially chosen as the side of the suspected bleeding source in all patients with sah , in patients wihout sah it was based on morphologic characteristics but not on speech dominance . factors for successful clipping of the contralateral aneurysm are as follows : the severity of cerebral edema , total length of the contralateral ( a1+m1 ) segments , configuration of the contralateral aneurysm , contralateral ica bifurcation angle , and projection of the contralateral aneurysm . compared with microscope in bmca aneurysms clipping via supraorbital keyhole approach , with the endoscope better visualization and ability to operate freely on the contralateral anterior cerebral artery , ica , m1 , and origin of m2 were achieved . the direction of the contralateral aneurysm is very important during bmca aneurysms clipping surgery via supraorbital keyhole approach . aneurysms pointing inferior are more difficult to clip from the contralateral approach because the mca regularly covers the neck region of the aneurysm and visualization of the perforating branches is poor . aneurysms pointing in lateral , anterior , or posterior directions are also more difficult to clip from the contralateral side because further inferior extension of the neck may be difficult to recognize . in these situations endoscope - controlled techniques have the following advantages : deep surgical field brightness can be increased and allow pathologic anatomy in a short distance to extend view angle and a clear descriptive . according to surgical need to change the depth the angle of the endoscope , and a different perspective of the endoscope can increase vision information , so as to achieve complete clipping the aneurysm , to avoid the occurrence of mistaken clipping . fully endoscope - controlled clipping intracranial aneurysms were rarely reported . in this study , we reported a case of bmca aneurysms endoscope - controlled clipping via unilateral supraorbital keyhole craniotomy . we believed that fully endoscope - controlled clipping bmca aneurysms was a practically feasible method , which combined advantages of both unilateral supraorbital keyhole approaches and endoscopic technology . because of the limited operative space and relative deep location , the surgery should be performed by experienced neurosurgeons . fully endoscope - controlled clipping bmca aneurysms may be a safe and effective application to increase the quality of treatment .

approximately 40% of patients with intracerebral hemorrhage die within 30 days , and the majority of survivors are left with severe disability4,11 ) . hematoma expansion occurs in up to 70% of patients who have ich documented by computed tomographic ( ct ) scanning performed within 3 hours after the onset of symptoms4,6,18 ) . other predictors of poor outcome include age , initial hematoma volume of hematoma , glasgow coma scale ( gcs ) , intraventricular hemorrhage ( ivh ) , warfarin use , and infratentorial hemorrhage10,14 ) . recently , several studies suggested that contrast extravasation on ct angiography ( cta ) is a crucial predictor of hematoma expansion and mortality7,8,12,30 ) . the presence of active contrast extravasation into the hematoma at the time of multi - detector ct angiography ( mdcta ) , the spot sign , is an indicator of active hemorrhage and has been associated with an increased risk of hematoma expansion and mortality in patients with ich6,7,8,12,30 ) . the purpose of this study was to retrospectively review cases of ich treated at our institution to determine if the cta ' spot sign ' predicts in - hospital mortality and clinical outcome at 3 months in patients with spontaneous ich . we used our institutional medical data search system to identify all adult patients who were admitted for medical treatment of ich between january 1 , 2008 and january 31 , 2012 . we conducted a retrospective review of all consecutive patients who were admitted to the department of neurosurgery . to be eligible for the study , patients with ich needed to meet the following inclusion criteria : 1 ) evidence of nontraumatic ich on a noncontrast ct ( ncct ) examination of the head , 2 ) evaluation with cta of the intracranial circulation within 24 hours of presentation , and 3 ) age between 18 and 80 years . the exclusion criteria were : 1 ) deep coma ( gcs 3 - 5 ) ; 2 ) brainstem hemorrhage ; 3 ) a history of previous intracerebral hemorrhage ; 4 ) pure intraventricular hemorrhage ; 5 ) secondary intracerebral hemorrhage such as arteriovenous malformation , moyamoya disease , tumor bleeding , and venous sinus thrombosis ; 5 ) incomplete standard ct protocol including ncct and mdcta ; 6 ) previous stroke history ; and 7 ) surgically treated patients . clinical data of patients with ich were collected by 2 neurosurgeons blinded to the radiological data and at the 90-day follow - up . the collected demographic and clinical variables included sex , age , alcohol and smoking use , history of hypertension , diabetes , liver disease ( liver cirrhosis and hepatocellular carcinoma ) , coronary heart disease , and medications ( antihypertensives , antiplatelet , and anticoagulation agents ) . the systolic , diastolic , and mean arterial blood pressures of patients were recorded . stroke severity on admission was evaluated by the gcs and national institutes of health stroke scale ( nihss ) . laboratory tests on admission included serum glucose , activated partial thromboplastin time ( aptt ) , and prothrombin time ( pt ) as expressed by the international normalized ratio ( inr ) . clinical outcomes were assessed by the modified rankin scale ( mrs ) and glasgow outcome scale ( gos ) on discharge and 90-day follow - up . ncct and mdcta acquisitions were performed according to standard departmental protocols on 16- or 64-section general electric helical ct scanners ( general electric medical systems , new york , usa ) . imaging was performed as follows : 1 ) initial and 24-hour follow - up ncct scans were performed using 4.5 mm contiguous axial sections from skull base to vertex parallel to the inferior orbitomeatal line . parameters for pre- and postcontrast ct were 120 kvp ; 340 ma ; 45 mm collimation ; 1 second / rotation ; and table speed of 15 mm / rotation . cta was performed following 0.7 ml / kg of iohexol ( 300 mg i / ml ; omnipa - que ; ge healthcare , piscataway , nj , usa ) injected intravenously at 4 ml / s rate via power injector through an intravenous line , using the following parameters : 120 kvp ; 240 mas ; section thickness , 1.25 mm ; and section - acquisition interval , 1 mm . all images were prospectively and independently reviewed on picture archiving and communication system ( pacs ) workstations by 2 neurosurgeons blinded to clinical data . the presence of contrast extravasation and spot sign score was determined according to the criteria from a prior study on mdcta source or reconstructed images8 ) . determination of initial and follow - up ich volumes was performed independently by investigators blinded to the initial ncct and follow - up ct . each reviewer measured the volume of hemorrhage in milliliters using the abc/2 method , where a is the greatest diameter of hemorrhage on the ct section with the largest area of hemorrhage , b is the diameter perpendicular ( 90 ) to a , and c is the number of sections with hemorrhage multiplied by the section thickness17 ) . c was calculated by a comparison of each ct section with hemorrhage , with the ct section demonstrating the largest area of hemorrhage on that scan . an increase of hematoma volume > 33% or > 12.5 ml was considered a hematoma expansion5,12,15 ) . the spot sign was defined according to four criteria : 1 ) serpiginous or spot - like appearance within the margin of a parenchymal hematoma without connection to an outside vessel ; 2 ) contrast density greater than 1.5 mm in diameter in at least one dimension ; 3 ) contrast density ( hounsfield units , hu ) at least double that of the background hematoma ; and 4 ) no hyperdensity at the corresponding location on non - contrast ct29 ) . statistical analyses were performed using the statistical package for the social sciences for microsoft windows ( version 12.0 ; spss inc . , chicago , il , usa ) . patients were classified according to survival versus fatality and good versus poor clinical outcomes ( mrs 0 - 2 versus 3 - 6 , gos 1 - 3 versus 4 - 5 ) on discharge and at 90-day follow - up . the relationships between spot sign with clinical outcomes and in - hospital mortality were examined by the chi - square test for categorical variables or mann - whitney u - test for continuous variables . the multivariate logistic regression analysis was repeated for the prediction of in - hospital mortality and poor outcome among survivors at 3-month follow - up including , as an additional variable , first the presence of any spot sign , and then mortality . the adjusted odds ratios ( ors ) and their 95% confidence intervals ( 95% cis ) were calculated . we used our institutional medical data search system to identify all adult patients who were admitted for medical treatment of ich between january 1 , 2008 and january 31 , 2012 . we conducted a retrospective review of all consecutive patients who were admitted to the department of neurosurgery . to be eligible for the study , patients with ich needed to meet the following inclusion criteria : 1 ) evidence of nontraumatic ich on a noncontrast ct ( ncct ) examination of the head , 2 ) evaluation with cta of the intracranial circulation within 24 hours of presentation , and 3 ) age between 18 and 80 years . the exclusion criteria were : 1 ) deep coma ( gcs 3 - 5 ) ; 2 ) brainstem hemorrhage ; 3 ) a history of previous intracerebral hemorrhage ; 4 ) pure intraventricular hemorrhage ; 5 ) secondary intracerebral hemorrhage such as arteriovenous malformation , moyamoya disease , tumor bleeding , and venous sinus thrombosis ; 5 ) incomplete standard ct protocol including ncct and mdcta ; 6 ) previous stroke history ; and 7 ) surgically treated patients . clinical data of patients with ich were collected by 2 neurosurgeons blinded to the radiological data and at the 90-day follow - up . the collected demographic and clinical variables included sex , age , alcohol and smoking use , history of hypertension , diabetes , liver disease ( liver cirrhosis and hepatocellular carcinoma ) , coronary heart disease , and medications ( antihypertensives , antiplatelet , and anticoagulation agents ) . the systolic , diastolic , and mean arterial blood pressures of patients were recorded . stroke severity on admission was evaluated by the gcs and national institutes of health stroke scale ( nihss ) . laboratory tests on admission included serum glucose , activated partial thromboplastin time ( aptt ) , and prothrombin time ( pt ) as expressed by the international normalized ratio ( inr ) . clinical outcomes were assessed by the modified rankin scale ( mrs ) and glasgow outcome scale ( gos ) on discharge and 90-day follow - up . ncct and mdcta acquisitions were performed according to standard departmental protocols on 16- or 64-section general electric helical ct scanners ( general electric medical systems , new york , usa ) . imaging was performed as follows : 1 ) initial and 24-hour follow - up ncct scans were performed using 4.5 mm contiguous axial sections from skull base to vertex parallel to the inferior orbitomeatal line . parameters for pre- and postcontrast ct were 120 kvp ; 340 ma ; 45 mm collimation ; 1 second / rotation ; and table speed of 15 mm / rotation . cta was performed following 0.7 ml / kg of iohexol ( 300 mg i / ml ; omnipa - que ; ge healthcare , piscataway , nj , usa ) injected intravenously at 4 ml / s rate via power injector through an intravenous line , using the following parameters : 120 kvp ; 240 mas ; section thickness , 1.25 mm ; and section - acquisition interval , 1 mm . all images were prospectively and independently reviewed on picture archiving and communication system ( pacs ) workstations by 2 neurosurgeons blinded to clinical data . the presence of contrast extravasation and spot sign score was determined according to the criteria from a prior study on mdcta source or reconstructed images8 ) . determination of initial and follow - up ich volumes was performed independently by investigators blinded to the initial ncct and follow - up ct . each reviewer measured the volume of hemorrhage in milliliters using the abc/2 method , where a is the greatest diameter of hemorrhage on the ct section with the largest area of hemorrhage , b is the diameter perpendicular ( 90 ) to a , and c is the number of sections with hemorrhage multiplied by the section thickness17 ) . c was calculated by a comparison of each ct section with hemorrhage , with the ct section demonstrating the largest area of hemorrhage on that scan . an increase of hematoma volume > 33% or > 12.5 ml was considered a hematoma expansion5,12,15 ) . the spot sign was defined according to four criteria : 1 ) serpiginous or spot - like appearance within the margin of a parenchymal hematoma without connection to an outside vessel ; 2 ) contrast density greater than 1.5 mm in diameter in at least one dimension ; 3 ) contrast density ( hounsfield units , hu ) at least double that of the background hematoma ; and 4 ) no hyperdensity at the corresponding location on non - contrast ct29 ) . statistical analyses were performed using the statistical package for the social sciences for microsoft windows ( version 12.0 ; spss inc . , patients were classified according to survival versus fatality and good versus poor clinical outcomes ( mrs 0 - 2 versus 3 - 6 , gos 1 - 3 versus 4 - 5 ) on discharge and at 90-day follow - up . the relationships between spot sign with clinical outcomes and in - hospital mortality were examined by the chi - square test for categorical variables or mann - whitney u - test for continuous variables . the multivariate logistic regression analysis was repeated for the prediction of in - hospital mortality and poor outcome among survivors at 3-month follow - up including , as an additional variable , first the presence of any spot sign , and then mortality . the adjusted odds ratios ( ors ) and their 95% confidence intervals ( 95% cis ) were calculated . from january 1 , 2008 until january 31 , 2012 , a total of 227 patients presented to the department of neurosurgery with spontaneous ich on ncct and were evaluated with mdcta of the intracranial circulation within 24 hours of admission ( fig . forty patients were excluded from the primary analysis for the following reasons : 20 patients were treated with surgical evacuation before follow - up ct ; 10 patients died before follow - up ct , and 10 patients did not have a follow - up ct for unknown reasons . a total of 187 patients met our inclusion criteria , with a mean age of 60.4514.49 years ( median 60.45 years , range 19 - 80 years ) . the median time from emergency department admission to mdcta evaluation was 1.33 hours ( mean 2.5 hours , range 0.25 - 8 hours ) , and median length of hospital stay was 14 days ( mean 17.72 days , range 2 - 95 days ) . cta demonstrated 61 cta spot sign - positive patients ( 61/187 ; 32.6% ) and 126 patients without the spot sign ( 126/187 ; 67.4% ) ( fig . ich was deep , lobar , or within the posterior fossa in 46 ( 34.6% ) , 120 ( 64.2% ) , and 21 ( 11.2% ) patients , respectively . follow - up results demonstrated 47 patients ( 25.1% ) with clinically important hematoma growth ; 35 of these demonstrated spot sign ( 74.46% ) on the initial cta ( table 1 ) ( fig . patients with clopidogrel use were more likely to have spot sign ( p=0.006 ) , but the small sample size ( n=11 ) was a limiting factor . univariate analyses demonstrated the spot sign ( p<0.001 ) , and clopidogrel use ( p= 0.001 ) were associated with hematoma expansion , whereas a history of hypertension , diabetes mellitus , antiplatelet use , anticoagulants , pt / aptt , inr , mean arterial blood pressure ( mabp ) 120 mm hg , in - hospital stay , and glucose 8.3 mmol / l had no association with hematoma expansion . hematoma expansion occurred significantly more frequently in patients with the spot sign than in those without ( p<0.001 ) . in multivariate logistic regression analysis , we found that the spot sign may play an important role indicating the presence of volume expansion ( or 5.010 ; 95% ci 1.993 - 12.599 ; p=0.001 ) , mrs ( or 7.706 ; 95% ci 1.021 - 7.169 ; p=0.045 ) , and in - hospital mortality ( or 8.870 ; 95% ci 2.554 - 30.804 ; p=0.001 ) ( table 2 ) . the associations between clinical , laboratory , and imaging variables and 90-day outcomes are shown in table 3 . the predictors of poor clinical outcome at 90-day follow - up include gcs , nihss , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , mabp , prothrombin time ( pt ) , inr , ivh , ivh volume , ich location , ich volume , hematoma expansion , spot sign , and treatment modality ( table 3 ) . multivariate logistic regression analysis identified predictors of poor outcome ; we found that hematoma location ( or 2.258 ; 95% ci 1.190 - 4.284 ; p=0.013 ) , spot sign ( or 3.883 ; 95% ci 1.467 - 10.275 ; p= 0.006 ) , ivh ( or 2.994 ; 95% ci 1.295 - 6.922 ; p=0.010 ) were independent predictors of poor outcome ( table 4 ) . in - hospital mortality was 57.4% ( 35 of 61 ) in the cta spot - sign positive group versus 7.9% ( 10 of 126 ) in the cta spot - sign negative group . we found that presence of spot sign ( or 10.197 ; 95% ci 2.572 - 41.157 ; p=0.001 ) and presence of volume expansion ( or 11.832 ; 95% ci 2.591 - 54.034 ; p=0.001 ) were independent predictors for the in - hospital mortality of ich ( table 5 ) . mortality and unfavorable outcome rates were high in spot sign - positive and volume expansion - positive patients . positive predictive values from the previous studies varied considerably ( 24 - 77% , 77.78% in this study ) , whereas negative predictive values were lower ( 96 - 98% , 81.68% in this study ) ( table 6)12,16,30 ) . we found that the spot sign is the strongest significant predictor of hematoma volume expansion , poor outcome ( mrs > 2 ) , and increased in - hospital mortality . the spot sign is also associated with larger hemorrhage , and a more severe clinical presentation . most recently , demchuk et al.9 ) concluded that the cta spot sign is highly predictive of hematoma expansion , irrespective of hematoma expansion definition and for both intraparenchymal and intraventricular hemorrhage growth . the cta spot sign is associated with poor prognosis , high rates of early clinical deterioration and mortality , often occurring within days after onset9 ) . hematoma expansion occurs in up to 70% of patients with ich documented by ct performed within 3 hours after the onset of symptoms4,5,6 ) . ich accounts for 10% to 20% of all strokes and is more fatal and disabling than ischemic stroke or subarachnoid hemorrhage ( sah)24 ) . because of the rapid and severe devastation associated with ich , innovative treatments need to be developed and evaluated . major predictors of increased early mortality and adverse outcome during the acute phase of ich are hematoma expansion , ivh with obstructive hydrocephalus , and hyperglycemia3,5,20,28 ) . at least 38 - 70% of patients had > 33% growth in the volume of parenchymal hemorrhage during the first 24 hours after symptom onset5,6 ) . a previous study reported that the volume of ich is the strongest predictor of 30-day outcome for all locations of spontaneous ich18 ) . hematoma expansion is associated with early neurologic deterioration and is an independent predictor of poor outcome and increased morbidity4,20,26 ) . mayer et al.20 ) proposed that hematoma expansion is associated with dysregulation of hemostasis via inflammatory cascade activation and matrix metalloproteinase ( mmp ) overexpression , breakdown of the blood - brain barrier , a sudden increase in icp leading to local tissue distortion and disruption , and vascular engorgement due to reduced venous outflow . hematoma expansion can also result from an increased plasma concentration of cellular fibronectin ( c - fn ) and the inflammatory mediator interleukin-6 ( il-6)27 ) . treatments to restrict hematoma expansion include hemostatic therapy , cautious lowering of high bp , quick reversal of prior anticoagulation , and surgical evacuation22 ) . clinical trials to restrict hematoma expansion in ich and a meta - analysis have shown that the use of recombinant factor vii ( rfviia ) limits the extent of hematoma expansion in patients with non - coagulopathic ich13,19,20,31 ) . however , there was an increase in thromboembolic risk with no clear clinical benefit in unselected patients . reports from the intensive blood pressure reduction in acute cerebral haemorrhage trial ( interact ) and antihypertensive treatment of acute cerebral hemorrhage ( atach ) trials show that sbp reduction might restrict hematoma expansion in the hyperacute phase of ich1,2,25 ) . many previous studies reported that spot sign is an independent predictor of hematoma expansion and mortality7,8,12,16,23,28,29,30 ) . brott et al.5 ) found the majority of relevant growth of the hematoma volume on admission ct , to occur within 4 hours after symptom onset . this suggests that growth occurs early in the course of spontaneous intracerebral hemorrhage ( sich ) and that early ct scan repetition is warranted to detect it . the timing of surgical treatment for ich is a key benchmark for hematoma expansion prevention because treatment delays are associated with an increased risk of mortality . early prevention may be an ideal target for medical or surgical hemostatic therapy in patients who exhibit high average rates of hematoma expansion . the timing of surgical treatment varies widely because of patient factors ( delayed symptom recognition and presentation to medical attention ) , physician factors ( delayed diagnosis and referrals ) , resource availability ( rapid access to vascular imaging ) , and institutional / system - level factors related to operating room availability and staffing . in our study , all patients arrived at the hospital within 10 hours of the clinical event . however , hematoma expansion during referrals occurred in over 35% of patients . theoretically , early surgical treatment putatively reduces hematoma expansion , reduces mortality , and improves clinical outcome . a report from the surgical trial in intracerebral haemorrhage ( stich ) trial showed no overall benefit of early surgical clot evacuation compared with initial conservative treatment in patients with ich21 ) . however , a subgroup analysis showed a potential benefit for surgery in lobar ich within 1 cm of the cortical surface21 ) . guidelines from the american heart association and american stroke association suggest that indications of surgical intervention are as follows : 1 ) for most patients with ich , the usefulness of surgery is uncertain ; 2 ) patients with cerebellar hemorrhage who are deteriorating neurologically or who have brainstem compression and/or hydrocephalus from ventricular obstruction should undergo surgical removal of the hemorrhage as soon as possible ; 3 ) for patients presenting with lobar clots > 30 ml and within 1 cm of the surface , evacuation of supratentorial ich by standard craniotomy might be considered ; 4 ) the effectiveness of minimally invasive clot evacuation utilizing either stereotactic or endoscopic aspiration with or without thrombolytic usage is uncertain and is considered investigational22 ) . clinical trials that are tailored more individually with regard to patient characteristics and sich features are still necessary to elucidate the role of surgery . further clinical trials should include surgical versus medical treatment in spot sign - positive patients . the use of recombinant factor vii ( rfviia ) limits the extent of hematoma expansion in spot sign - positive patients . the number of 187 patients was relatively small , and the data set was heterogeneous with regard to time to scan . a prospective study with a larger number of cases is needed to confirm the association of spot sign to poor clinical outcomes . the spot sign is a strong independent predictor of hematoma expansion , mortality , and poor clinical outcome in primary ich . unfortunately , all measures to restrict hematoma expansion have so far failed to improve outcome in randomized controlled trials . in this study , we emphasized the importance of he - matoma expansion as a therapeutic target in both clinical practice and research . clinical trials of surgical interventions and hemostatic treatment , such as rfviia , should be done in ich patients with a positive spot sign on cta .

to report a case who had recurrence of macular hole retinal detachment ( mhrd ) after intravitreal ranibizumab injection ( ivr ) for the treatment of choroidal neovascularization ( cnv ) that arose from the damaged retinal pigment epithelium of the remaining macular hole ( mh ) edge , which had been successfully treated by pars plana vitrectomy ( ppv ) 15 years previously . a 67-year - old man with previous ppv for mhrd secondary to high myopia in the right eye had been under observation for 15 years after surgery . ppv with an additional internal limiting membrane peeling , removal of the cnv membrane and 20% sf6 gas tamponade was performed . one year after the last surgery , his right retina was attached and the mh was closed successfully . we propose that patients who undergo ivr should be carefully maintained and followed up for possible complications including the recurrence of mhrd . intravitreal anti - vascular endothelial growth factor ( vegf ) agent therapy for age - related macular degeneration ( amd ) , proliferative diabetic retinopathy ( pdr ) and neovascular glaucoma ( nvg ) is widely used . on the other hand , retinal pigment epithelium ( rpe ) tears [ 1 , 2 , 3 ] and worsening of tractional retinal detachment ( rd ) [ 4 , 5 , 6 ] have been reported as a result of the contractile forces of regressing neovascular membranes after intravitreal anti - vegf agent injection . however , the mechanism of construction of the fibrovascular membranes remains to be elucidated . in this report , we present a case who had recurrence of macular hole retinal detachment ( mhrd ) after intravitreal ranibizumab injection ( ivr ) for the treatment of choroidal neovascularization ( cnv ) that arose from the damaged rpe of the remaining macular hole ( mh ) edge . a 67-year - old man with previous pars plana vitrectomy ( ppv ) for mhrd secondary to high myopia in the right eye ( od ) had been under observation for 15 years after the surgery . slit lamp examination revealed pseudophakia in od and mature cataract in the left eye ( os ) . best corrected landolt ring chart visual acuity ( bcva ) was from 0.1 to 0.2 in od and 30 cm finger motion in os . the patient presented with sudden vision loss in od . on presentation , funduscopic examination revealed a disciform rpe atrophy formation at the bottom of the remaining mh and cnv development around the edge of mh from the superior to temporal side ( fig . his bvca was not worsening but his subjective symptom was getting worse day by day . funduscopic examination showed cnv contraction to the superior edge and recurrence of mhrd from the inferior edge of the remaining mh ( fig . emergency ppv with an additional internal limiting membrane peeling , removal of cnv and 20% sf6 gas tamponade was performed on the same day . extraction of cnv was performed under the patient 's written informed consent , the approval of the institutional review board in kobe kaisei hospital and the tenets of the declaration of helsinki . one year after the last surgery , his right retina was reattached and mh was closed successfully ( fig . 1c , e , h ) . rpe tears and worsening of tractional rd after intravitreal anti - vegf agent therapy have been reported [ 1 , 2 , 3 , 4 , 5 , 6 ] . several reports hypothesized that intravitreal anti - vegf agent therapy may cause severe contract force of the neovascular membrane followed by rpe tear in amd [ 1 , 2 , 3 ] and tractional rd in pdr [ 4 , 5 , 6 ] . it is possible that the contract force of the regressing cnv might be increased after ivr , thereby causing rd from the remaining mh edge in our case . in our case , we performed emergency ppv with cnv removal to fix the detached retina because the visual acuity in os was bad and the mhrd in od was expanding . for the treatment of cnv , it might be one of the options to continue ivr because ivr is thought less invasive compared with cnv removal during ppv . but if we perform additional ivr first , it is very important to make the cnv inactive before the following ppv because ivr itself may cause recurrence of mhrd due to the contract force of the cnv again . we must follow - up this patient for at least another month after the injection to confirm the effect of ivr . unfortunately , we had not a moment to lose in this case . we performed ppv with cnv extraction and achieved both retinal reattachment and the quieting effect of the cnv . in summary , we propose that patients who undergo ivr should be carefully maintained and followed up for possible complications including the recurrence of mhrd .

artifacts or simulated diseases are self - inflicted conditions caused by various means and for different purposes . disease simulation can be motivated , among other things , by illegal purposes , to escape from civil duties or prison sentences , for example , or to exploit specific situations in order to receive a range of benefits . in such cases , we report the case of a 42-year - old woman who , for 3 consecutive years , showed religious stigmata on the forehead and on the dorsal surface of hands and feet immediately before easter . lesions showed an acute onset , manifested as erosions and ulcerations , and healed a few days after easter . stigmata were immediately made public and every year faithful and curious people went in procession to the house of the patient , offering different kinds of gifts . after intervention of the police authority in the early days of the third episode , the patient and her family repented of their actions . cutaneous lesions healed in few days and similar events did not occur during the following 2 years . regardless of personal beliefs , the possibility that stigmata could be self - inflicted for illegal or profit purposes should always be considered . the issue about self - inflicted skin lesions ( sisl ) has been critically discussed in a recent position paper of the european society for dermatology and psychiatry . sisl are divided into 2 main groups : the first group is represented by syndromes associated with a denied or hidden pathological behavior , with ( malingering ) or without ( factitious disorders ) external incentives ; the second group is represented by syndromes associated with a nondenied and nonhidden pathological behavior ( compulsive and impulsive disorders ) . our case of religious stigmata belongs to the first group of sisl , associated with external incentives and profit purposes . a 42-year - old woman , originally from a small town in calabria ( south of italy ) , showed , for the third consecutive year , cutaneous lesions during easter holidays . such lesions appeared every year on the forehead and on the dorsal surface of hands and feet ( figs . 13 ) . lesions suddenly become manifest on holy monday and healed spontaneously within a few weeks . these alleged stigmata were made public , causing a steady flow of devotees and curious onlookers to the patient 's house , offering gifts of various kinds . the year before the local ecclesiastical and police authorities had become interested in the singular event , but legal or religious actions were not undertaken as the stigmata had rapidly disappeared some days after easter . the patient came to our attention on the third day of clinical manifestations of the third year , accompanied by her general practitioner . at physical examination , a dozen mildly exudative erosions were present on the forehead , along the hairline , measuring 0.5 cm in diameter , regularly spaced 1 cm from each other . on the dorsal surface of the hands , halfway between carpus and metacarpus , an irregularly round erythematous - ulcerated lesion , swollen and moderately bleeding , was noticeable . similar erythematous - ulcerated elements ( measuring 1.5 1 cm ) involved symmetrically the central dorsum of the feet . no lesions were present on wrists , palms , soles , or elsewhere . during consultation the patient was silent ; she gave no specifics on time and mode of onset of lesions , and did not complain of any subjective symptoms . she also showed a wary and absent attitude , occasionally speaking in a detached manner about her religious faith . the psychiatric consultation , which had been refused at the time of the previous episodes , described a hysterical personality with attention - seeking behavior and excessive need for approval of her family . moreover , her general practitioner reported us that the intervention of the police , the day before , had stopped the people pilgrimage to the house and had changed the attitude of the patient and her relatives ; they showed fear and discretion , diminished the episode , and argued that most likely it would not have occurred again . the patient refused hospitalization but promised she would return for follow - up , but actually never showed up . her general practitioner later reported us that lesions were already healing 3 days after our consultation , with complete healing after 3 weeks . most of the time disease simulations are due to psychiatric problems , such as psychosis and personality disorders . the intrinsic motive of these lesions is a request for family and society attention , as a reaction to difficulties or adverse situations with involuntary cutaneous somatization , masochism with physical pain , and guilt or spiritual responses . as showed by the literature , cases of unconscious simulators , mainly women with chronic cutaneous diseases , belong to this group . such patients always deny self - induced illness and seem unconcerned about the disease . however , disease simulation can be motivated by illegal purposes , for example , to escape from specific types of duties ( military duty , draft ) , to avoid detention , and to acquire benefits in an occupational environment ( sickness leave extension , recognition as an occupational disease , higher class of disability ) . in these cases , simulators are aware of their actions and purposes . in our long - term practice , we have directly observed 46 patients affected by sisl with illegal purposes in occupational environments . these patients , overwhelmingly male , are affected by morphologically bizarre lesions , more so than those provoked by unaware pathomimia patients . besides hands and forearms , unusual areas are involved and the most peculiar and unlikely causative agents are implicated . the certainty of conscious simulation , suspected by a positive history for job uneasiness , by lesions particular morphology , and by repeated diagnostic tests negativity , came from patients admission after repeated friendly and confidential consultations . herein we report a rare case of religious stigmata . affected sites , morphology , acute onset , and as a matter of fact , in our case hands and feet dorsum were affected without the involvement of palms and soles . moreover , in religious texts , the originally described sites of upper limb involvement are the wrist and not the hands . stigmata were also immediately made public by the patient and her family in this case . our hypothesis was indirectly confirmed by quick resolution of the dermatosis and no recurrence of the lesions during the following 2 years . moreover , the patient 's absent attitude , as though she was living in her inner world , could also underlie a hysterical personality that could be easily exploited by her relatives . skin lesions were probably caused by metal objects , by a mechanism of scraping ( on the forehead ) , or overheating ( most likely on the dorsal surface of hands and feet ) ; however , the use of caustic chemicals can not be excluded . differential diagnosis with dermatologic conditions , when singularly considering a lesion among those affecting hands and feet , included contact dermatitis , mycosis , and infective and noninfective granulomatosis ; contact dermatitis to a hair accessory such as a frontlet or headband ( perhaps with spaced metallic inserts ) could have been responsible for the hairline manifestations . however , when assessing the clinical presentation as a whole , together with patient history , we believe that a differential diagnosis is not needed , as there is no single dermatologic condition that could justify the morphology , localization , symmetry , and evolution of such lesions . to our knowledge , the topic of malingering religious stigmatization has rarely been treated in the medical literature . kluger and cribier thoroughly discussed the subject in light of modern medicine , and a few other cases similar to our own have been reported . in particular , a case of a 42-year - old man with a 5-month history of palmar ulcers at different healing stages has been described . during consultation the patient theatrically explained how he had been blessed by god with christ 's stigmata . however , the patient 's partner revealed that such lesions were autoinflicted with a knife . another case of a 23-year - old male patient with stigmata on the hands has been reported in a small town of sicily . he showed some erosion on the dorsal surface of hands , and small bullous lesions arranged to create the word previously , other odd events , promoted as divine , had occurred in the house of the young man ( weeping of an effigy depicting the madonna , weeping and bleeding of a jesus christ 's picture , lugubrious sounds at night ) . accordingly , a prayer hall in the garage of the house had been set up , complete of an altar and iconography of the stations of the cross . the patient later turned out to be hysterical , with an easily swayed personality , and easily manipulated by relatives and acquaintances for profit aim . lifschutz observed a 10-year - old black baptist girl during a 3-week period immediately preceding easter sunday . psychiatric examination did not evidence psychological alterations except an indifference toward bleeding and auditory hallucinations of religious nature . the issue concerning religious stigmata has always been of particular interest , given the complexity of interpretation and discussion . stigmatized people present bleeding lesions in 5 distinct body sites : hands and feet because of nails ( not on wrists , as evidenced by the shroud of turin ) , the ribs in relation to the thrust of the spear , the back ( site of flagellation and pressure of the cross ) , and the forehead because of the crown of thorns . besides the visible ones , invisible stigmata , such as intense pain affecting the above - mentioned sites , should be considered . the latter could precede or represent the outcome of visible stigmata , but may also be isolated , without any means to ascertain their presence . saint francis of assisi ( 11821226 ) was the first person to receive stigmata , unless saint paul implied that he had them when he wrote , saint francis developed the stigmata during an ecstatic vision in 1224 ; these were described 2 years later in his first biography . the saint francis 's stigmata were immediately acknowledged by the papacy , despite minor opposition among the clergy , which accused the saint of heresy . kluger and cribier reported 3 other cases of autoinflicted stigmata , which dated back to the xiii century . during the following centuries , > 300 cases of stigmatization were described , most of which were fervent roman catholics . in 1908 , imbert - gourbeyre found that of the 321 people with stigmata described afterwards saint francis , 41 were men and 280 were women during their reproductive years . they mainly came from italy ( 229 cases ) , and then france , spain , germany , belgium , portugal , switzerland , holland , hungary , and peru . among the most famous stigmatized of the xix century , louise lateam ( 18501883 ) stands out , a belgian girl who bled every friday except for 2 from 1868 to 1883 ; she was visited by various eminent belgian doctors . also threse neumann ( 18981962 ) , a german peasant girl , developed the stigmata in 1926 during an ecstatic vision ; her hands and feet bled regularly on fridays until her death . finally saint pio of petralcina ( 18871968 ) had his hands bleeding continually from 1918 ; however , the lesions faded at the time of his death . as shown by the above - mentioned data , stigmata have been manifesting for centuries in the context of catholic europe . in the usa , in the xx century , stigmata were reported among other religious contexts ( e.g. , muslim ascetics developed lesions in the same sites of injuries endured by the prophet in his battles ) . however , few people with stigmata have been beatified or canonized , and the catholic church does not allow for mandatory association between stigmata and sanctity , declining the conventional belief that stigmata represent a supernatural event , and are granted by god as a sign of piety . a proclaim by pope urban viii instructs catholic writers to attribute a purely human nature to this phenomenon . the question of the etiological interpretation of stigmata remains controversial ; as a matter of fact , there is no reason to presume that all stigmata should have the same etiology . excluding ascertained cases of voluntary self - harm for malingering , as our case and few others ( table 2 ) , stigmatization is generally referred to as unconscious self - harm during hysteria , autosuggestion , and hypnotism ; these are all characteristic factors that can be associated with mystical delirium . cases of religious stigmata due to self - harm for malingering purposes . according to various authors , however , cases of inexplicable stigmata with spontaneous onset and absence of external triggering factors have been described . discrete exceptional clinical entities , not fully understood in their pathogenesis , could possibly constitute the underlying condition to some of these cases , for example , autoerythrocyte sensitization syndrome ( gardner diamond syndrome , or psychogenic purpura ) , vicarious menstruation ( various organs possibly affected ) , hematidrosis ( blood cells in sweat ) , and hemolacria ( secondary to ophthalmic conditions ) . such conditions usually affect young women , although often presenting variable features of psychosomatic personality , and are certainly considered natural . given all the earlier discussion , religious stigmata are progressively shifting from medieval mysteries to distinct psychiatric and psychosomatic disorders . in studying stigmata , however , we should always consider the possibility that they could be self - inflicted for illegal and/or profit purposes . women of catholic faith are mainly affected . in the context of a state of trance , bleeding lesions appear , overlapping those suffered by jesus christ , on hands and feet ( not on wrists , as evidenced by the shroud of turin in relation to the spikes ) , sometimes on the ribs ( in relation to the thrust of the spear ) , or on the back ( site of flagellation ) , and less often on the forehead ( in relation to the crown of thorns ) or on the shoulders ( in relation to the pressure areas of the cross ) . in conclusion , regardless of personal beliefs , we should always consider the possibility that stigmata , whose study does typify the fascinating relation between physical , psychological and spiritual phenomena , could be self - inflicted for illegal and/or profit purposes .

india is facing a shift from undernutrition to overnutrition,1 later producing obesity and its aftermaths like type 2 diabetes mellitus ( t2 dm ) . there is alarming rise of t2 dm in india,2 a country with an ethnic predisposition for it . insulin resistance proceeded by obesity is the link between t2 dm and cardiovascular deaths3 and at least the first event can be monitored and prevented primarily to stop this chain of progression . however , there is a lack of awareness regarding obesity and optimum body composition which are not given importance as due as glycemic control measurement and maintenance in case of known t2 dm subjects.4 body mass index ( bmi ) , though defining obesity , falls short of qualitative inference of body composition5 while the modern imaging techniques that overcomes this deficit are far from reach in our community to the most . bioelectric impedance analysis ( bia ) provides an objective , cost - effective method of qualitative and quantitative body composition analysis with proven efficacy in our population6 with inference about visceral fat ( vf ) that is a risk factor itself and a negative affecter for glycemic control.7 however , once diagnosed what is the effect of these parameters on body composition especially body fat , remains a question . we conducted this study to find the impact of glycemic control using glucose triad on bia derived parameters of body composition in terms of both quantity and quality . we conducted a cross - sectional observational study from january 2013 to april 2014 in clinical research lab , physiology department of our medical college . sample size of 78 for current population of city 6,00,000 and prevalence of t2 dm 7.33% in urban area of our state8 gave us 90% confidence interval keeping margin for error 5% as calculated by sample size calculator software graphpad in stat 3 software ( demo version free software of graphpad software , inc . following approval from the institutional review board and informed consent from participants , the study was carried out in under treatment ambulatory sedentary type 2 diabetics . subjects were recruited from medicine outpatient department ( opd ) of a tertiary care teaching hospital attached to our medical college and from private opds . about 78 type 2 diabetics ( 44 males and 34 females ) were undertaken in the age group 3080 years , living sedentary life , not taking insulin , taking regular medicines , and having a recent investigation for glycemic or lipidemic control . to make the sample heterogeneous , we included patients with and without hypertension , with and without statin therapy , with or without family history of type 2 diabetes , coming from various socioeconomic statuses so as to make a fairly representative sample of the population . to evaluate glycemic control of the type 2 diabetics subjects underwent measurement of ( 1 ) fasting blood sugar ( fbs ) and postprandial blood sugar ( pp2bs ) done by god - pod method ( 2 ) glycosylated hemoglobin ( hba1c ) done by immunoturbidimetry method . these tests were done as a recent report by fully auto analyzer i lab-650/miura , a-1004 at naac certified biochemistry department of our college using standard sops . we defined glycemic control as per criteria laid by american diabetes association 2014,9 and good glycemic control was defined as ( 1 ) hba1c 7 g % , ( 2 ) fbs 126 mg% , and ( 3 ) pp2bs 180 mg % . subjects were divided into two groups based on these criteria into those with good or poor glycemic control . subjects meeting inclusion and exclusion criteria were undertaken for the study with initial assessment in the form of personal history , medical history , anthropometric measurement , and recent reports of glycemic controls including , fbs , pp2bs , and hba1c , and lipidemic control . after entering age , gender , and height taken by stadiometer subject was allowed to stand on the instrument after its calibration . a digital , portable , noninvasive instrument omron karada scan ( model hbf-510 , china ) working on the principle of tetra polar bioelectrical impedance analysis was used that passes electric current of 500 a at frequency 5 khz to scan the whole body to derive regional body composition . we enrolled ambulatory outdoor patients only and took the reading in the morning so as to avoid dehydration10 that otherwise would affect the accuracy of this method . for qualitative analysis , we defined standard norms as-1 ( bmi 252 ) ( vf 103 ) total body fat ( tbf ) and skeletal muscle mass as per standard guidelines.11 the data were transferred on excel spreadsheet , and descriptive analysis was expressed as a mean standard deviation . we evaluated the difference between of these body composition parameters among groups based on glycemic control quantitatively by student 's t - test and qualitative risk calculation by odds ratio using defined cutoff norms of body composition parameters . any observed difference was considered statistically significant with p < 0.05 . the data were transferred on excel spreadsheet , and descriptive analysis was expressed as a mean standard deviation . we evaluated the difference between of these body composition parameters among groups based on glycemic control quantitatively by student 's t - test and qualitative risk calculation by odds ratio using defined cutoff norms of body composition parameters . any observed difference was considered statistically significant with p < 0.05 . table 1 shows baseline data of study group reflecting the participation of both sexes , average duration of type 2 diabetes 7.5 years , high average bmi , good lipidemic control , and poor glycemic control with respect to hba1c . base line data of case group under study table 2 shows direct quantitative correlation between values of bia derived parameters of body composition with means of glycemic control , namely , hba1c , fbs , and pp2bs reflecting that subjects , regardless the glycemic status , showed the high - fat low muscle mass pattern of body composition which is slightly more so in case of poor glycemics , yet statistically insignificant in most instances . quantitative comparison of parameters of body fat distribution among groups based on glycemic control ( defined by therapeutic goals ada guidelines 2014 ) to get a clear picture for correlation , we defined cutoff points for variables , namely , bmi , vf , tbf , skeletal muscle mass , and tried to calculate odds risk ratio for their abnormality owing to exposure to uncontrolled blood sugar for all three measures of glycemic triad . this qualitative comparison showed that there was small , inconsistent , and insignificant odds risk of poor glycemic control ( hba1c , fbs , and pp2bs ) on parameters of body composition with none bearing adequate strength of association [ table 3 ] . qualitative comparison of parameters of body fat distribution ( defined by standard cut off norms ) among groups based on glycemic control ( defined by therapeutic goals ada guidelines 2014 ) obesity has officially been declared a disease by american medical association in 2013 and india is no different to other countries when it comes to seriousness of its increasing magnitude that too with unique attributes . india shares one - third of the total burden of t2dm4 worldwide that is further compounded by obesity doubling the cost of its management.12 for given bmi , south asians have greater adiposity and visceral and ectopic adipose tissue accumulation.13 few studies have revealed more adverse fat distribution at bmi > 21 kg / m2 in south asians as compared with caucasians in whom considerable dyslipidemia and dysglycemia are unseen until bmi exceeds 30 kg / m2.14 in previous studies , in type 2 diabetics of our region , we found poor glycemic control and high prevalence of many preventable risk factor.15 we also found that type 2 diabetics have more ectopic fat on expense of skeletal muscle that persists even after matching by weight or bmi , both quantitatively and qualitatively.16 with this propensity , it seems quite worthful to know body composition and body fat , in particular , both quantitatively and qualitatively in not only high - risk obese subjects but also in type 2 diabetics with respect to current glycemic control . t2 dm patients of our study had high bmi , vf , subcutaneous fat ( sf ) , tbf , and lesser muscle mass that is attributed to high mean age , average duration of disease 7.5 years , poor glycemic control and sedentary lifestyle , apart from disease itself . we found no effect of glycemic control on parameters of body fat distribution measured indirectly by bia in ambulatory sedentary t2 dm subjects , for almost all three of glycemic triad in terms of both quality and quantity with exception of tf - fbs and sf - hba1c . this is similar to a recently reported study.17 correction of hyperglycemia decreases the risk of microvascular complications but macrovascular complications to a lesser extent that otherwise represent the primary cause of mortality with heart attacks and stroke accounting for around 80% of all deaths.31819 most diabetic patients undergo regular scrutiny of glycemic and lipidemic control , and when it comes to body composition , bmi is perhaps only option offered to the most . however , bmi falls short of many qualitative inferences especially vf , which can be objectively measured by bia . obesity is the primary event , and one of the risk factor for t2 dm and once t2 dm ensues all measures turn to secondary in this regard . t2 dm , a multifaceted metabolic derangement , is more a disease of abnormally altered lipid metabolism than merely that of carbohydrates.20 it is evident now that it is not the disordered glucose metabolism but rather the chronic elevation of free fatty acid that is the culprit for t2dm.21 diabetic patients target blood sugar and blood lipid control at the same time neglecting the deranged pattern of body composition in the form of increased ectopic fat at expense of protein that is associated with higher cardiovascular comorbidities.22 vf has now proven to bring about insulin resistance that leads to diabetes and there are evidence based on bariatric surgery23 and exercise intervention studies17 that reduced vf improves glycemic status as well as insulin resistance . however , the situation is further compounded by the facts that treatment for diabetes itself causes adiposity,24 preventive pharmacotherapy has the least effect on optimizing body composition,25 mild to moderate exercise affects body fat little,26 and indians are most vulnerable to obesity.4 there are fallacies while relating glycemic status and body compositions such as effect of glycemic variability making current glycemic status not completely reliable,27 poor glycemic control in indian diabetics28 especially with regard to hba1c , use of subjective methods such as waist - hip ratio,29 no glycemic threshold for micro or macrovascular complications of t2dm30 and ethnic vulnerability of indians for obesity - related complications.4 obesity is a disease and not a choice.4 prevention of weight gain is one of the therapeutic goals for t2 dm patients.31 many rely solely on statins which in the absence of other lifestyle interventions are ineffective to optimize body composition as shown by our another work.32 we also found current lipidemic control to affect body fat only insignificantly . weight reduction is good not only for improving glycemic control but also for reduction of cardiovascular risk.33 weight regain is very common , and weight loss is difficult to maintain.34 subjects can be motivated for optimum body composition by regular bia scan for body fat and self - monitoring can definitely be reinforced . one can be motivated for lifestyle modifications such as diet plans , exercise , and smoking cessations that can serve as measures of secondary prevention achieved by self - care in t2 dm subjects and measures of primary prevention in those at risk by self - awareness , in both the cases , helping to fight against modern epidemic of obesity and its aftermath t2 dm . this study has few limitations such as its cross - sectional nature , small sample size , presence of risk factors which can not be eliminated and the method which is based on a predictive formula , tending to underestimate body fat . however , it showed that in t2 dm patients , abnormality of body composition especially vf has no correlation with glycemic status , hence requiring special attention for knowing , targeting , and achieving optimum body composition using simple methods such as bia to make sure that prevention turns better than cure . we found no correlation of current glycemic status with abnormally elevated ectopic fat and reduced muscle mass in under treatment sedentary type 2 diabetics , suggesting the need for qualitative body composition by methods like bia , optimizing it by lifestyle modifications , and maintaining it to reduce adverse outcomes in an attempt to fight against obesity .

even though both diseases are rare and may display features of clonality , it is possible that the present concurrence should be regarded as fortuitous . the true prevalence of pediatric mastocytosis is not known , but estimated to be similar to adult mastocytosis and around 10 in 100.000 1 . whereas adult mastocytosis is almost always a systemic disease with a clonal proliferation of neoplastic mcs caused by the somatic kit d816v mutation , pediatric mastocytosis is mostly limited to the skin and 8090% of affected children exhibit a spontaneous remission before puberty 2 . the kit d816v mutation can only be detected in 2535% of affected children 3 , however , mutations in kit outside exon 17 are reportedly prevalent , indicating that pediatric mastocytosis in many cases is a clonal disease associated with somatic mutations in kit 4 . the prevalence of lyp have been estimated to be 1.21.9 per million in adults 6 . pediatric cases have rarely been described and reportedly associated with atopy in up to 60% 7 , as also found in the present case . clinically it is characterized by recurrent crops of erythematous papules and nodules that resolve after weeks to months , leaving atrophic scars . potential differential diagnoses in childhood include insect bites , pityriasis lichenoides ( pl ) , and cd30positive , anaplastic large tcell lymphoma among others . differentiating lyp from skin lesions caused by insects is usually straightforward clinically as well as histologically . skin lesions in pl are usually smaller and of shorter duration than lyp and histologically , lichenoid infiltrates without cd30positive t lymphocytes are usually seen , but in atypical cases a clear distinction between lyp and pl may be difficult to make . an important and sometimes difficult differential diagnosis of lyp , type c is cd30positive anaplastic large tcell lymphoma , and in rare cases the histological distinction is impossible and clinical course decisive . in adult patients a malignant transformation has been described with mycosis fungoides , hodgkin lymphoma and cd30 positive largecell lymphoma being most common . in pediatric lyp the absolute risk for transformation is estimated to be very low , however , has rarely been reported , for example , by nijsten and colleagues who described development of nonhodgkin lymphoma in 3 of 35 children with lyp 7 . thus , a followup in line with adults is recommended 5 . in the present case we did not detect evidence of associations between the preexisting mastocytosis and development of lyp . cellular lineages involved differ , and we did not detect histologic signs of mc infiltration in the lyp skin lesions . in summary , we report a unique case of a child with concomitant lyp and cutaneous mastocytosis . both diseases are rare and have to our knowledge not been reported together before . even though it seems probable that the two conditions occurred together by chance further studies are needed to elucidate if any associations between lyp and mastocytosis exist .

first described in 1980 , 1 percutaneous endoscopic gastrostomy ( peg ) is the most common minimally invasive method for establishing long - term enteral feeding access . pneumoperitoneum is relatively common after peg , reported in 8.6 % to 56 % of cases , and usually self - limited , however , it may be associated with post - procedural bloating , discomfort , and pain 3 4 5 6 7 . air is thought to leak into the peritoneum from full insufflation of the stomach during the between trochar needle insertion and final positioning of the gastrostomy tube 6 . in the absence of other clinical evidence of peritonitis , conservative management for post - peg pneumoperitoneum however pneumoperitoneum can also be a sign of perforation and can lead to imaging , hospitalization , and even exploratory laparotomy after peg placement 8 . until recently , ambient air was the default insufflation agent for endoscopy given its ease of use , wide availability , and lack of additional costs . however , carbon dioxide is increasingly being used for both routine and therapeutic endoscopic procedures , including colonoscopy and ercp , with consistent evidence for reduced post - procedural bloating and pain due to more rapid reabsorption 9 10 11 12 . in addition , if perforation occurs , capnoperitoneum is less pronounced and clinically detrimental due to the rapid reabsorption and its use is recommended for therapeutic endoscopic procedures 13 . the only previous study evaluating the role of co2 insufflation during peg placement used placement of multiple gastropexies prior to standard pull - through technique 7 . in that study , co2 compared to ambient air was associated with less frequent post - procedural pneumoperitoneum and a reduction in small bowel distention , but pain and discomfort were not evaluated . the aim of our study was to compare co2 vs. ambient air insufflation during peg placement without the use of gastropexy for differences in the rate of post - procedural pneumoperitoneum as well as abdominal distention , pain , and bloating . this was a single - center , randomized , double - blind prospective study conducted at the university of utah hospital . the study was approved by the institutional review board and informed consent was obtained from patients or their surrogate medical decision - makers . consecutive patients undergoing peg were asked to participate from june 2012 to june 2014 . once enrolled , patients were randomized to co2 or ambient air using a computerized random number generator . the study coordinator ( kc ) instructed endoscopy technicians on which insufflation agent should be used . endoscopists and radiologists were not present for this step and remained blinded to the agent being used . for patients in whom co2 insufflation was used , it was delivered with the co2efficient endoscopic insufflator ( bracco diagnostics , monroe township , nj ) on the managed flow setting at 3.4 l / min . for ambient air , an evis exera iii clv-190 was used on the medium air flow setting ( 0.68 l / min ) . a 20f peg ( endovive safety kit , boston scientific , natick , ma ) , gastropexy was not performed and the peg tube y - port adapter was temporarily opened immediately after placement to decompress the stomach . procedure time , sedation doses , and any immediate complications were recorded from each procedure . no local anesthetic was used during any procedures , and there was no additional post - procedural sedation administered prior to evaluation for post - placement pain / bloating . post - peg pneumoperitoneum was evaluated with a left lateral decubitus abdominal x - ray obtained within 30 minutes of endoscopic placement . all images were interpreted by an experienced gastrointestinal radiologist ( ds ) who was blinded to the insufflation agent used . when present , the degree of pneumoperitoneum was subjectively graded as small , moderate or large . as a surrogate for distention , abdominal circumference was measured at the level of the umbilicus with the patient supine immediately prior to peg placement and then again within 30 minutes of the procedure . after the procedure , patients were asked to complete two visual analog scales ( vas ) , one for general post - procedural pain and another specifically assessing abdominal bloating . each patient s chart was reviewed 30 days after the procedure to evaluate for any delayed complications . statistical analysis was performed using stata version 10 ( statacorp lp , college station , tx ) . a fisher s exact test was used to evaluate for a significant difference in the rate of pneumoperitoneum between groups , while student s t - test was used to evaluate for difference in abdominal girth and for differences in vas scores for pain and bloating . a - priori statistical power analysis was completed and , assuming post - peg pneumoperitoneum rates of 30 % for ambient air and 10 % for the co2 group , we calculated that 15 patients were needed in each arm to reach the standard statistical power threshold of 0.8 . a total of 35 subjects undergoing peg placement were enrolled during the study period , of whom 17 patients were randomized to co2 and 18 patients were randomized to ambient air . n = 18 ) , neurogenic dysphagia ( n = 13 ) , malnutrition ( > 10 % loss of ideal body weight ) ( n = 1 ) , and high aspiration risk ( n = 3 ) . the preponderance of patients with als is because the university of utah is the regional referral center for the disease . there were no statistically significant differences between groups , although there was a trend toward more females with als in the ambient air group . there were no subsequent hospitalizations or reported complications within 30 days of peg placement for any patient . peg , percutaneous endoscopic gastrostomy ; bmi , body mass index ; als , amyotrophic lateral sclerosis post - procedural left lateral decubitus x - rays were obtained within 30 minutes of peg placement except in three patients in the co2 group and three patients in the ambient air group , leaving 14 patients randomized to co2 and 15 patients to ambient air . one patient had an error in order entry that led to prolonged delay in imaging , and the other five patients declined to participate in additional imaging due to postoperative discomfort or excessive sedation within the 30-minute window . outcomes of peg placement using co2 vs ambient air are summarized in table 2 . pneumoperitoneum was significantly less frequent when using co2 , with post - procedure pneumoperitoneum in 2/14 ( 14.3 % ) compared to 8/15 ( 53.3 % ) using ambient air ( p = 0.05 ) . there was no significant difference in the radiographic size of pneumoperitoneum between groups ( p = 0.65 , table 2 ) . there was no significant difference in pre- and post - procedure abdominal circumference , with a mean change in waist measurement of 3.1 3.1 cm in the co2 group vs. 3.0 2.5 cm in the ambient air group ( p = 0.85 ) . peg , percutaneous endoscopic gastrostomy ; vas , visual analog scale patients with end - stage als often had difficulty interacting with the vas , especially after procedural sedation , and there was a high dropout in this aspect of the study , limiting its value and statistical power . six patients in the co2 arm ( 3 with als ) and nine patients in the ambient air arm ( 4 with als ) declined or were unable to complete the vas . only 11 patients in the co2 and nine patients in the ambient air group completed vas . there were no significant differences in scores for abdominal pain ( 4.3 vs. 2.2 , p = 0.12 ) or for abdominal bloating ( 2.8 vs. 1.9 , p = 0.46 ) . this study showed a significant decrease in the frequency of post - procedural pneumoperitoneum when using co2 . we report a rate of post - peg pneumoperitoneum of 53 % when using ambient air compared to only 14.3 % with co2 . this is the first study to measure and evaluate subjective measures of pain and bloating after peg using co2 insufflation , however , it did not find a significant difference in abdominal girth or in patient - reported pain or bloating between co2 and ambient air insufflation . incidence of post - peg pneumoperitoneum using ambient air varies widely in published reports ( 8.6 % -56 % ) 4 5 6 7 . this is likely due to numerous differences in study design , including timing and sensitivity of imaging modalities and differences in gastrostomy technique . the lowest reported rate ( 8.6 % ) was a retrospective study of standard chest x - rays not specifically obtained to evaluate for post - peg pneumoperitoneum . other studies obtaining plain upright and ap chest x - rays ( rate 20.9 % ) or plain abdominal films obtained in a variety of positions ( rate 38 % ) performed within 3 hours of peg reported intermediate rates of pneumoperitoneum 4 6 . abdominal computed tomography ( ct ) , the most sensitive imaging modality for identifying intraperitoneal free air , was used in two studies . one series obtaining ct 24 hours after peg reported a rate of 26.7 % , however , as previously noted , this study used gastropexy 7 . in the second series , in which abdominal ct scans were obtained from 1 hour to 9 days after gastrostomy , the rate was 56 % , but this study was performed by interventional radiology under push - technique with dilators , which likely increased the incidence of pneumoperitoneum 5 . our method of using a fairly sensitive technique , left lateral decubitus film , obtained soon ( within 30 minutes ) after peg placement likely resulted in our incidence of pneumoperitoneum air being at the higher end of the reported range . our study is the second report to evaluate co2 insufflation during peg and confirms previous results demonstrating that co2 use significantly reduces the rate of post - peg pneumoperitoneum 7 . our rate of capnoperitoneum was 14.3 % , substantially higher than the previous study of 58 patients , which reported a rate of 0 % . first , our left lateral decubitus x - rays were obtained 30 minutes after placement , compared to their ct scans , which were obtained 24 hours after placement . it is very likely that relatively small amounts of co2 within the peritoneum will be completely resorbed within 24 hours ; therefore , our timelier imaging likely gives a more accurate rate of the immediate post - procedural capnoperitoneum rate . second , the previous study used three gastropexies during peg placement prior to trocar insertion and pull peg placement . we found higher rates of pneumoperitoneum when either ambient air ( 53 % vs. 27 % ) or co2 ( 14 % vs. 0 % ) was used , suggesting that gastropexy reduces air leakage into the peritoneum during peg regardless of insufflation agent . gastropexy is common during peg placement in japan , but is not widely used in the united states , making our results more relevant when gastropexies are not used . we found no difference between ambient air and co2 in change of abdominal girth before and after the procedure . previous studies of upper and lower endoscopy using similar methodologies for measuring abdominal girth did report differences in abdominal girth when insufflating with co2 vs. ambient air 11 14 . our patients were vented immediately after peg placement , which may have contributed to the lack of difference in abdominal girth between groups . in addition , peg procedures are significantly shorter than ercp and colonoscopy with less volume of insufflation agent , thus this method may not be sensitive enough to detect relatively smaller differences in intraluminal bowel distention . in the previously cited study evaluating co2 vs. ambient air for peg , kub was used to measure bowel distention after peg and less distension in the small bowel was reported with co2 but no difference in distention was reported in the colon 7 . our method of abdominal girth measurement may not be sensitive enough to have detected differences in distention isolated to the small bowel . this is the first study to evaluate for differences in patient - reported pain and bloating between co2 and ambient air during peg placement . we found no difference in vas scores in pain or bloating , although several patients were unable to participate in the evaluation shortly after placement due to severe neurologic impairment ( end - stage als ) or persistent sedation . other endoscopic procedures , including colonoscopy and endoscopic retrograde cholangiopancreatography ( ercp ) , have been associated with significant improvement in post - procedural bloating and pain when using co2 . the fact that peg placement did not seem to show a benefit in symptoms may be explained by the shorter duration of peg and the lower total volume of insufflation introduced , as well as the fact that patients undergoing peg have more comorbidities and may be less prone to notice minor differences in symptoms . our study also confirms that post - peg pneumoperitoneum by itself is a clinically benign finding . we agree that in the absence of other clinical signs of perforation ( e. g. peritoneal signs , subcutaneous emphysema , or sepsis ) , post - peg pneumoperitoneum does not require intervention or warrant further workup . however , the lower incidence and more rapid resolution of benign pneumoperitoneum with co2 may help prevent confusion regarding the need for additional imaging , evaluation , and even laparotomy to rule out perforation . it enrolled fewer patients than previous series , and not all of them participated in the post - procedure x - ray to evaluate for pneumoperitoneum . however , equal numbers of patients were lost in each group and we did reach our predetermined enrollment size for the primary outcome as calculated by a - priori power analysis . not all patients were willing or physically capable of participating in vas quantification of post - procedural pain and bloating . this aspect of the study was limited by the smaller numbers of patients participating in vas scoring , however , there was no dramatic difference in vas scores . a slight difference in symptoms between co2 and ambient air is unlikely to have clinical relevance , given that peg patients are often fairly debilitated with high rates of comorbid disease . plain x - ray is a less sensitive modality than abdominal ct for demonstrating pneumoperitoneum . we compensated , however , by obtaining images within 30 minutes of peg placement , using left lateral decubitus films , and having a single highly experienced gastrointestinal radiologist interpret all the films . measurement of abdominal girth at the level of the umbilicus may not be sensitive or reliable enough to detect smaller differences in bowel distention or pneumoperitoneum , however , we felt that obtaining abdominal cts in patients without other clinical signs or symptoms was not clinically indicated and was prohibitively expensive . ambient air insufflation produces noise compared to the relatively silent pressurized co2 system , which may have affected blinding of the endoscopist during peg placement . however , we believe this effect was minimal because our suction vacuum produces significantly more noise than other insufflation systems , and the radiologist and assistant measuring study variables were not present during actual endoscopy . co2 significantly reduces the frequency of post - procedural pneumoperitoneum compared to ambient air as an insufflation agent during peg placement . there were no significant differences in measured abdominal distension or patient - reported symptoms of abdominal bloating or pain . reduction in the incidence and duration of pneumoperitoneum may help prevent benign pneumoperitoneum being misconstrued as a sign of perforation , preventing unnecessary evaluation , imaging , and even surgical intervention . carbon dioxide appears to be safe and effective and may be the insufflation agent of choice for peg placement .

quinupristin / dalfopristin ( synercid ) , a combination of two different streptogramins antibiotics , has a bactericidal effect by inhibiting the bacterial protein synthesis of ribosome . it is currently used in infections caused by vancomycin - resistant enterococcus faecium ( vref ) , when no other active antibacterial agent is available . the known adverse effects of quinupristin / dalfopristin are peripheral vasculitis , arthralgia , myalgia , nausea , diarrhea , vomiting and skin papules . sweet s syndrome , or acute febrile neutrophilic dermatosis , is characterized by painful skin papules , and plaques , fever and peripheral blood leukocytosis , with dermal neutrophilic infiltration on a skin biopsy . drug - induced sweet s syndrome has a temporal relationship between the drug ingestion and clinical presentation , or a temporally - related recurrence after oral rechallenge , with temporally - related resolution of the lesions following drug withdrawal or on treatment with systemic corticosteroids . the authors , having encountered a patient , who was the first to our knowledge , developed sweet s syndrome after administration of quinupristin / dalfopristin , herein report on the case with a review of the english literature relating to the other adverse effects of the drug and the clinical features of the syndrome . a 63-year - old woman , with end stage renal disease , receiving hemodialysis three times a week , was hospitalized for acute subdural hemorrhage . on the 30th day following her admission , she had a high fever of 39c and complained of costovertebral angle ( cva ) tenderness on physical examination . vref was isolated in a urine culture , but with blood cultures yielding no growth of microorganism . the patient was treated with quinupristin / dalfopristin ( synercid , 7.5 mg / kg , intravenously , through a central venous catheter , every 8 hours ) . on the 1st day of the quinupristin / dalfopristin treatment , her body temperature was normalized and the cva tenderness subsided , but she complained of nausea , vomiting , and myalgia . on the 2nd day of treatment with the drug , she complained of severe myalgia , arthralgia and general weakness , and presented tender erythematous 2 cm - sized papules on the palms of her hands ( figure 1a ) . on the 3rd day of treatment the skin papules , which started on the palm , spread to the face , chest and neck , and progressed to maculopapular plaques or pustules ( figure 1b ) . the white blood cell count was 20,600 cells / mm ( neutrophils , 84% ) , the hemoglobin was 7.0 g / dl , the platelet count was 209,000 cells / mm , but her liver enzymes were within normal limits . a punch biopsy of a skin lesion on her palm was performed , which showed intra- and sub - corneal pustular formation , with epidermal leukocytosis ( figure 2 ) . there was no evidence of vasculitis , necrosis or infections caused by bacteria , fungus or mycobacteria , in the biopsy specimen . she had received no other drugs that could have caused the drug - induced sweet s syndrome . the quinupristin / dalfopristin treatment was stopped , after which , the severity of these side effects were rapidly relieved , and the pain from the skin lesions subsided without analgesics . in 1964 , sweet described a new disease , in eight female patients , which was characterized by the acute onset of fever , leukocytosis , and tender skin plaques infiltrated by neutrophils : he named the disorder acute febrile neutrophilic dermatosis . in as many as 50 percent of patients , sweet s syndrome is associated with a variety of underlying diseases : malignancy , bacterial and viral infections , drugs ( furosemide , hydralazine , oral contraceptives , minocycline , nitrofurantoin , trimethoprim - sulfamethoxazole and granulocyte - stimulating factor , autoimmune and collagen vascular diseases ( rheumatoid arthritis , systemic lupus erythematosus , mixed connective tissue disease , hashimoto thyroiditis , sjogren s syndrome ) , inflammatory bowel disease ( chron s disease , ulcerative colitis ) and pregnancy . the pathogenesis of sweet s syndrome is still unknown , but it is currently thought that cytokine dysregulation , a type 3-hypersensitivity reaction or a t cell - dependent cellular immune reaction is involved . diagnostic criteria have been proposed for sweet s syndrome , with a patient having to meet two major and two minor criteria for the diagnosis to be confirmed . one major criterion is the abrupt onset of typical cutaneous lesions , with the other being a histopathology compatible with sweet s syndrome . there are four minor criteria antecedent fever or infection ; accompanying fever , arthralgia , conjunctivitis , or underlying malignancy ; leukocytosis and good response to systemic corticosteriods , but no response to antibiotics . the cutaneous lesions are characterized by tender or painful erythematous or violaceous plaques or nodules . they are most often found on the face , neck and upper extremities , especially on the dorsum of the hands , but they can occur anywhere . the diagnostic histopathologic feature of sweet s syndrome is nodular and dense perivascular neutrophilic infiltration , with no evidence of vasculitis . drug - induced sweet s syndrome is found to be resolved promptly , without recurrences , on discontinuation of medication , or with systemic steroid therapy . in patients with drug - induced sweet s syndrome , it is difficult to definitively determine whether the cause of the dermatosis is drug - induced , or related to an underlying condition . two useful criteria for establishing a diagnosis of drug - induced sweet s syndrome are a temporal relationship between the drug administration and the clinical presentation , and between drug withdrawal and resolution of the disease . quinupristin / dalfopristin is a novel parenteral antimicrobial agent , consisting of two different semisynthetic , water - soluble , streptogramins antibiotics , quinupristin ( 30% ) and dalfopristin ( 70% ) . they are synergistic , and usually bactericidal against gram - positive , selected gram - negative and some anaerobic organisms , and is an effective therapy for serious infections due to vref . it has a unique mechanism of action that involves bacterial cell entry , by diffusion of the drug to the bacterial ribosome , followed by the formation of stable dalfopristin - ribosome - quinupristin complexes . consequently , this combination of the two components may prevent the appearance of strains resistant to either of the components arthralgia , without objective signs of articulopathy , and myalgia are the most commonly reported adverse events , and the leading causes of treatment discontinuation . gastrointestinal adverse events ( nausea , vomiting , and diarrhea ) and skin rash have also been documented . all theses side - effects are reversible on discontinuing the drug , and its overall tolerability is good . patients receiving at least one intravenous infusion of quinupristin / dalfopristin have been known to experience local venous intolerability ( pain , burning , inflammation , and thrombophlebitis ) . laboratory findings of the adverse events are the increases in alkaline phosphatase , aminotransferases and -glutamine transferase , as well as decreases in sodium , hemoglobin , haematocrit , platelet count and red blood cell count the drug is primarily excreted by the liver ( 75% ) , with a small portion of the unchanged ( 1519% ) form eliminated by the kidneys . a recent study indicates that no formal dosage reduction would appear necessary in patients with severe chronic renal impairment or hepatic dysfunction , although there is a tendency for slower clearance of unchanged dalfopristin and active quinupristin derivatives . however , the effect of a dose reduction or an increase in the dosing interval , on the pharmacokinetics of quinupristin/ dalfopristin in these patients requires further study , so no recommendations can be made at this time . our patient , who received quinupristin / dalfopristin treatment , initially presented with arthralgia , myalgia , nausea , vomiting and fever , with maculopapular plaques that progressed to pustules . after discontinuation of the drug , the systemic adverse events were quickly resolved , and the cutaneous lesions cleared within 7 days . the patient receive no systemic corticosteroids . although unproved , it is not inconceivable that in this patient the quinupristin / dalfopristin was involved in the clinical and histological reactions . the drug has never been implicated in the literature as a causative agent of sweet s syndrome before . additional studies are warranted to determine the mechanism of sweet s syndrome associated with the drug .

human immunodeficiency virus ( hiv ) infection has become associated with decreased morbidity and mortality since the advent of highly active antiretroviral therapy ( haart ) in western countries,1 non - western asian settings,2,3 and sub - saharan regions.4 however , older age , hiv infection itself , antiretroviral therapy ( art ) , hiv - related inflammation , current cd4 t cell count , nadir cd4 t cell count < 350 cells / mm , and traditional risk factors for cardiovascular disease ( smoking , obesity , physical inactivity , excess alcohol intake ) substantially increase the risk of cardiovascular disease , including hypertension , stroke , coronary artery disease , peripheral artery disease , dyslipidemia , diabetes mellitus , metabolic syndrome , and lipodystrophy.517 the occurrence of chronic haart - associated noncommunicable diseases is attributable to complex molecular mechanisms , including the direct impact of haart on lipid metabolism , endothelial and adipocyte cell function , and mitochondria.18 experts have dichotomized the nadir cd4 t cell count into < 350 cells / mm versus 350 cells / mm to define the optimal time to start art.1921 changes in cd4 counts from very low levels to at least 200 cells / mm reduce the frequency of progression of hiv disease.22 in hiv patients of black african descent , psychosocial determinants , including new charismatic religion , significantly delay progression of the disease to acquired immune deficiency syndrome ( aids).2325 in developed countries , rates of type 2 diabetes mellitus and insulin resistance12 have been increasing among hiv - infected patients after the introduction of haart . in these settings , the incidence of type 2 diabetes among patients with hiv disease receiving protease inhibitors and other antiretroviral molecules is estimated to be 1%13%.7,1315 causes of type 2 diabetes in hiv - infected patients are complex , and include the effects of hiv , which is a chronic inflammatory and insulin - resistant condition in itself , genetics , cigarette smoking , physical inactivity , obesity , and aging.16 however , in sub - saharan africa , with the exception of south africa,4 art in general and protease inhibitors in particular are not readily available at present . only 6% of hiv - infected patients have had access to generic art since 2004.26 in the democratic republic of congo , combination art is defined as at least triple therapy including two nucleoside reverse transcriptase inhibitors ( stavudine + lamivudine or zidovudine + lamivudine ) plus one non - nucleoside reverse transcriptase inhibitor ( nevirapine or efavirenz ) . there are many reports of the increasing prevalence , incidence , and risk factors for type 2 diabetes in general populations,2731 but we do not have data about the disease in hiv patients from the democratic republic of congo who have progressed to aids . therefore , the objective of this study was to determine the cross - sectional association between a number of sociodemographic factors and art , and the incidence and predictors of type 2 diabetes among central africans with hiv disease . this study was of mixed design and included both a cross - sectional analysis of baseline sociodemographic , cardiometabolic , and hiv characteristics ( cd4 , nadir cd4 , cd8 , hiv-1 rna , and combination art ) and a longitudinal analysis of nadir cd4 count and the incidence of new - onset type 2 diabetes mellitus while on combination art at 3-monthly intervals during the study . the study included two groups of patients ( exposed or not exposed to art ) followed up at the teaching hospital of kinshasa university medical school , democratic republic of congo , between january and december 2008 . the institutional research board / ethics committee of kinshasa university approved the study protocol , which complied with the declaration of helsinki . a total of at least 87 hiv - infected patients aged 18 years was needed in each group to give the study sufficient power to detect such an increase in risk ( smaller incidence in one group = 0.01 ; higher incidence in the other group = 0.11 ; two - sided type i error , = 0.10 ; power 80% , http://www.epibiostat.ucsf.edu/dcr/ ) . patients were followed prospectively during their regular visits to outpatient or inpatient services . at enrolment ( baseline in 2004 ) and at least every 3 months thereafter , standardized data collection forms were completed to provide information about gender , age , family and personal history of diabetes , cigarette smoking , alcohol intake , physical inactivity , marital status , religion , anxiety ( stress , death of spouse , lack of social support ) , socioeconomic status , blood pressure , treatment for diabetes , lipid - lowering therapy , antihypertensive therapy , and any other sexually transmitted infections . at baseline , a comprehensive evaluation was performed , including measurements of weight , height , body mass index ( bmi , kg / height in m ) , waist circumference , hip circumference , systolic blood pressure , diastolic blood pressure , cd4 cell count , cd8 cell count , viral load ( hiv rna level ) , total cholesterol , cumulative exposure to art ( months ) , and duration of hiv-1 infection ( years ) . at enrolment and during each longitudinal visit , an oral glucose tolerance test was performed after an 814-hour fast overnight , with a standard oral load of 75 mg glucose in 250300 ml water , and glucose determination on arrival and after 2 hours using hexokinase - glucose-6 phosphate dehydrogenase ( biomerieux , marcy letoile , france ) and an autoanalyzer ( hospitex diagnostics , florence , italy ) at the same central laboratory at lomo medical , kinshasa limete democratic republic of congo ( variation coefficient of intratest and intertest for plasma glucose being 1.3% and 1.6% , respectively ) . all the information collected was transported into a standardized format and merged into a central data set at the clinical epidemiology unit , lomo medical center , kinshasa limete , democratic republic of congo . all incidental cases of type 2 diabetes were reported to the study coordinating office at lomo medical center for central validation and coding . all new - onset cases were identified in one of two ways , ie , a definite diagnosis if fasting plasma glucose was 126 mg / dl ( 7.0 mmol / l ) and/or 2h - glucose post load plasma glucose was 200 mg / dl ( 11.1 mmol / l ) . diabetes mellitus was defined according to world health organization ( who ) criteria , ie , fasting plasma glucose > 126 mg / dl ( 7.0 mmol / l ) measured on two consecutive occasions . high and low socioeconomic status were defined according to our previous population - based survey.8 hiv disease staging was defined according to who criteria11 and centers for disease control and prevention classification.12 nutritional status included underweight ( bmi < 18.5 ) , ideal weight ( bmi 18.524.9 ) , overweight ( bmi 2529.9 ) , and obesity ( bmi 30 ) . therefore , bmi 25 ( overweight / obesity ) defined total obesity versus abdominal obesity with waist circumference 94 cm , a congolese population - specific cutoff for both men and women as a surrogate of clinical insulin resistance,13 and peripheral obesity with hip circumference 97 cm , a congolese population - specific cutoff for both men and women.14 lifestyle factors ( cigarette smoking , physical inactivity , excessive alcohol intake ) were defined according to the who stepwise approach.15 religion was divided in groups , ie , one comprising the new charismatic religion imported from african - american churches ( miracles , positive coping ) and another group comprising the traditional churches ( catholic , protestant , muslim ) . in the democratic republic of congo , art strategies include a combination of stavudine + lamivudine , zidovudine + lamivudine + nevirapine , or efavirenz . high cardiovascular risk was defined as elevated rates of cigarette smoking , alcohol intake , abdominal obesity , overweight / total obesity , and peripheral obesity . data were expressed as the mean standard deviation for continuous variables or as proportions ( % ) for qualitative variables . means and proportions were compared using the student s t - test and the chi - square test , respectively . trends of proportions were examined using linear - by - linear chi - square tests ( p for trend ) . in univariate analysis , the relative risk of type 2 diabetes and its 95% confidence interval ( ci ) was calculated ( mantel haenszel test ) . in multivariate analysis , the cox regression model was used to quantify the relationship ( hazards ratio and its 95% ci ) between exposure to art , other univariate potential risk factors , and the risk of type 2 diabetes after adjustment for possible differences in the participant cardiometabolic risk profile . data were regarded as statistically significant when the p - value was < 0.05 ( two - tailed test ) . all statistical analyses were performed using statistical package for the social sciences ( spss ) software for windows version 13.0 ( ibm corporation , armonk , ny ) . diabetes mellitus was defined according to world health organization ( who ) criteria , ie , fasting plasma glucose > 126 mg / dl ( 7.0 mmol / l ) measured on two consecutive occasions . high and low socioeconomic status were defined according to our previous population - based survey.8 hiv disease staging was defined according to who criteria11 and centers for disease control and prevention classification.12 nutritional status included underweight ( bmi < 18.5 ) , ideal weight ( bmi 18.524.9 ) , overweight ( bmi 2529.9 ) , and obesity ( bmi 30 ) . therefore , bmi 25 ( overweight / obesity ) defined total obesity versus abdominal obesity with waist circumference 94 cm , a congolese population - specific cutoff for both men and women as a surrogate of clinical insulin resistance,13 and peripheral obesity with hip circumference 97 cm , a congolese population - specific cutoff for both men and women.14 lifestyle factors ( cigarette smoking , physical inactivity , excessive alcohol intake ) were defined according to the who stepwise approach.15 religion was divided in groups , ie , one comprising the new charismatic religion imported from african - american churches ( miracles , positive coping ) and another group comprising the traditional churches ( catholic , protestant , muslim ) . in the democratic republic of congo , art strategies include a combination of stavudine + lamivudine , zidovudine + lamivudine + nevirapine , or efavirenz . high cardiovascular risk was defined as elevated rates of cigarette smoking , alcohol intake , abdominal obesity , overweight / total obesity , and peripheral obesity . data were expressed as the mean standard deviation for continuous variables or as proportions ( % ) for qualitative variables . means and proportions were compared using the student s t - test and the chi - square test , respectively . trends of proportions were examined using linear - by - linear chi - square tests ( p for trend ) . in univariate analysis , the relative risk of type 2 diabetes and its 95% confidence interval ( ci ) was calculated ( mantel haenszel test ) . in multivariate analysis , the cox regression model was used to quantify the relationship ( hazards ratio and its 95% ci ) between exposure to art , other univariate potential risk factors , and the risk of type 2 diabetes after adjustment for possible differences in the participant cardiometabolic risk profile . data were regarded as statistically significant when the p - value was < 0.05 ( two - tailed test ) . all statistical analyses were performed using statistical package for the social sciences ( spss ) software for windows version 13.0 ( ibm corporation , armonk , ny ) . of 174 eligible patients with hiv disease , 102 patients with complete data ( response rate 58.6% ) participated in this longitudinal cohort . the study population included 49 patients infected with hiv-1 and exposure to combination art and 53 patients infected with hiv-1 not on combination art . the mean known duration of hiv-1 infection was 3.8 1.8 years in the study population and in each exposure group . tables 1 and 2 show the baseline characteristics of the hiv patients at entry into the study and stratified by their art exposure . patients on and not on combination art were comparable ( p > 0.05 ) for general baseline characteristics . figure 1 shows the distribution of the study population at study entry according to nutritional status and art exposure . it shows unexpectedly low rates of underweight , with two thirds of participants being of ideal normal weight and one third being overweight or obese . rates of abdominal obesity ( 39.6% , n = 40 ) and peripheral obesity ( 36.3% , n = 37 ) were also high among these patients with hiv disease . abdominal and peripheral obesity were more prevalent in patients with art exposure ( peripheral obesity in 55.1% , p = 0.002 ; abdominal obesity in 63.3% , p < 0.01 ) than in patients without art exposure ( peripheral obesity in 18.9% , and abdominal obesity in 17% ) . paradoxically , abdominal obesity / clinical insulin resistance was present in patients who were underweight , of normal weight , and with overweight / total obesity ( figure 2 ) . at 3-month follow - up , changes in nadir cd4 count from less than to more than 200/mm occurred in 45 patients on combination art ( 98.8% ) and in three patients not on combination art ( 5.7% ) , and the difference was statistically significant ( p < 0.0001 ) . significantly lower rates of anxiety , smoking , and excess alcohol intake but higher values for hiv duration and baseline cd4 counts were present in patients following new charismatic religion than those following a traditional religion ( p < 0.0001 , table 3 ) . overall , five of 102 patients developed new - onset type 2 diabetes ( rate 4.9% or 9.8 per 1000 person - years ) . male gender , following a traditional religion , an increase in nadir cd4 count , exposure to art , and single status were identified as significant univariate risk factors for incident cases of type 2 diabetes ( table 4 ) . however , after multivariate analysis and adjustment for gender , arv exposure and marital status , only changes in increased nadir cd4 count and following a traditional religion independently predicted onset of type 2 diabetes ( table 5 ) . the data show that these african hiv patients are at high cardiovascular risk because of unexpectedly elevated rates of total obesity , smoking , excess alcohol intake , abdominal obesity , peripheral obesity , and physical inactivity . physical inactivity and social stigma may explain the high rates of obesity in our study because there was not significant association between anthropometry , total cholesterol , and exposure to art at the entry . this study demonstrated that before introduction of art in 2004 , no cases of diabetes mellitus were reported among these hiv - infected patients . hiv infection on its own was previously thought to be a significant protective factor against the onset of diabetes . however , although protease inhibitors were not available to these patients , the present study has shown an increased risk of type 2 diabetes amongst them , as reported elsewhere.7,1315,3234 the estimated incidence rate of 4.9% ( 9.8 per 1000 person - years ) of type 2 diabetes in the present cohort remains within the interval of 1%13% reported in africa.1315,35 in western countries , cardiometabolic effects related to protease inhibitors are well known in hiv patients.35,36 however , the incidence was lower than the 1747 per 1000 person - years of incident cases of diabetes in the multicenter aids cohort study.36 ethnicity , sample size , criteria used to define diabetes ( a single elevated fasting blood glucose measurement ) , treatment with protease inhibitors , demographic composition ( older age ) , and higher bmi may explain the differences in incidence rates for diabetes among hiv patients and across the continents . in the us , protease inhibitors increase the likelihood of diabetes developing with age in african - americans infected with hiv.34 our results are consistent with those of other cohorts showing a similar relationship between exposure to art without protease inhibitors,16,35,36 older age , male gender,35 and incidence of type 2 diabetes . indeed , older age , cigarette smoking , and excess alcohol intake are well established risk factors for diabetes among hiv patients.35 single status may constitute a stress / anxiety with hypersympathicotonia , which is known to determine hyperglycemia . as in people not infected with hiv , age is an important risk factor for both diabetes and coronary heart disease in people with hiv in developed countries.37 the nucleoside reverse transcriptase inhibitors may contribute to a higher risk of diabetes in central africans with hiv via different mechanisms . there was a significant association between abdominal obesity ( a surrogate for clinical insulin resistance in this ethnic group ) , peripheral obesity , and exposure to art . although the mechanisms of action by which protease inhibitors and nucleoside analogs trigger diabetes is not fully understood , most of the hypothesized pathophysiology implicates the insulin resistance pathway16,17 rather than insulin secretion.38 it is important to highlight that exposure to art had no significant effect on cd4 counts in this study . this means that other factors , such as effective control of hiv - related opportunistic infections , appropriate diet ( vitamins ) , and reduced stress may increase the cd4 count,39 including the nadir cd4 count during the early stages of art in hiv patients with cd4 counts > 350 cells / mm and during monitoring of treatment , ie , clinical pharmacovigilance . indeed , treatment - nave hiv patients in the same area of kinshasa and practicing new charismatic religion imported from the us receive more social support , have lower rates of depression and stress , and are less likely to progress to aids than their counterparts practicing the more traditional religions.2325,39 this may explain the higher risk for new - onset type 2 diabetes in hiv patients with new traditional religion . indeed , many studies suggest that hiv itself is an inflammatory and insulin - resistant state which can precipitate overt diabetes mellitus,40 although there has been a case report of a 52-year - old african man whose type 2 diabetes resolved when viral replication was suppressed with protease inhibitor - based art.41 advanced hiv disease may be associated with an increase in counter - regulatory hormones whereby excess free fatty acids in the circulation reduce insulin sensitivity , hypercortisolism with further stress response as hiv disease progresses,42 growth hormone,43 high tumor necrosis factor alpha levels,44 interleukin-6,45 and hiv protein vpr acting as a glucocorticoid agonist.46 consistent with the obesity epidemic in the general population,29,47,48 the present study shows similarly increasing rates of obesity in hiv patients during the hiv pandemic . indeed , hiv patients exposed to art may be living longer49,50 and experiencing a lower frequency of aids - related wasting syndrome.5154 the clinical implication of the present study is that we should screen aggressively for diabetes in patients with hiv disease to avoid this double burden of disease in africa . art - related obesity and type 2 diabetes are becoming increasing problems in central africans with hiv disease . a relative increase in nadir cd4 cell count and traditional religion practices appear to be the strongest independent predictors of risk of type 2 diabetes .

lindau ( vhl ) disease.1 rchs are hamartomatous malformations most commonly detected in patients between ages 25 and 30 years.1 they are located juxtapapillary in 17% of cases.1 in general , juxtapapillary rchs ( jrchs ) are treated if they are progressive or if they affect visual acuity ( va).2 herewith , we report a typical case of progressive jrch that was successfully treated with a single injection of intravitreal ranibizumab ( ivr ) . a 44-year - old man , known to have vhl disease was referred to our ophthalmology clinic for routine fundus examination in 2008 . the patient had chronic renal failure after he underwent bilateral partial nephrectomy for renal cell carcinoma , and he also had spinal cord hemangioma diagnosed by spinal magnetic resonance imaging . on examination , results of slit - lamp examination of both eyes and fundus examination of the right eye were normal . fundoscopy of the left eye revealed an exophytic juxtapapillary capillary hemangioma along the nasal margin of the disc . no serous retinal detachment or exudates fluorescein angiography of the left eye showed early and late hyperfluorescence of the lesion with no leakage ( figure 2a ) . a computerized humphrey automated perimeter ( humphrey - zeiss , san leandro , ca , usa ) ( program 30 - 2 ) threshold test for visual field ( vf ) was normal in both eyes . no action was undertaken , and the patient was asked to return to the clinic yearly for follow - up . on subsequent follow - up visits , we noticed a progressive increase in the size of the hemangioma , which in 2012 reached a diameter double that of the optic disc ( figure 1d ) . also , some exudates and a minimal late leakage were observed in the peripapillary region on fluorescein angiography ( figure 2c ) . due to the rapid increase in size of the jrch , we decided to treat the patient with ivr . after detailed discussion with the patient , informed consent was received and ivr ( 0.5 mg ranibizumab ; lucentis , genentech , inc , san francisco , ca , usa ) was performed as an off - label treatment . three weeks after a single injection of ivr , vascular channels within the hemangioma became less prominent . six months after the single ivr , fundus examination showed a fibrotic , yellow - white appearance as a result of tumor scarring , and only few vascular channels could be seen within the hemangioma ( figure 3 ) . fluorescein angiography revealed a decrease in the vessels within the hemangioma and a decrease in late leakage ( figure 2d ) . during all after ivr , there was no further increase in hemangioma size , no significant systemic or ocular adverse events associated with ivr were noted , and no additional injections were needed . most rchs show slow progression over time , but a small percentage may regress spontaneously . if rchs enlarge over time and are left untreated , risk of exudation and subretinal fluid accumulation , cystoid macular edema , and retinal detachment increase , which can lead to progressive visual loss.2 the goal in the treatment of rch is preservation of va and the vf without destruction of the function of the retina around the tumor . treatment depends on the size and location of the hemangioma and the clinical manifestations.2 treatment strategies for jrch change from observation to laser coagulation , radiation therapy , transpupillary thermotherapy , vitreoretinal surgery , and photodynamic therapy ( pdt ) . in addition , juxtapapillary lesion therapies are often recalcitrant to treatment and the visual outcome of rch after treatment varies.3 in general , observation is chosen as the initial management of jrch , as they behave differently from the peripheral type of rch . some jrchs have been noted to remain stable for years.1,2 the treatment of these lesions should only be undertaken if vision is reduced or if there is lesion progression , as treatment usually leads to significant reduction in acuity due to adverse effects on the optic nerve and major retinal vessels.2 transpupillary thermoplasty has been used in the treatment of two reported cases of jrch that resulted in complete fibrosis , although one case developed optic atrophy.1 laser photocoagulation applied to the neural tissue surrounding the tumor can cause permanent scotomas.2 external radiation therapy has been shown to be effective in the treatment of optic disc hemangiomas,2 but due to the posterior location of the tumor and its proximity to optic nerve , all these approaches are risky and can result in permanent va deterioration or scotomas . pdt was shown to be effective in causing fibrosis and involution of the primary angioma , but its use on the peripapillary area is limited by vaso - occlusive effects . for larger tumors , verteporfin may only be activated on the surface of the tumor , and the reactive oxygen species may not cause closure of deeper tumor vessels.4 recent studies indicate that rch may depend on vascular endothelial growth factor ( vegf ) , as patients with vhl disease who present with rch have elevated ocular levels of anti - vegf.5 anti - vegf therapy has been reported to reduce vascular permeability by altering the balance of vasoactive cytokines like nitric oxide and endothelin-1 or by directly altering endothelial tight junction proteins.6 it is postulated that excessive accumulation of hypoxia - induced factor in the neoplastic stromal cells of rch leads to the production of other angiogenic factors that are able to maintain and promote the growth of primary hemangiomas.7 many cases have been reported in which jrchs were treated successfully with systemic administration of the anti - vegf ( vegf - receptor inhibitor ) su5416 , with a decrease in macular edema.79 it was believed that this treatment decreased the vasopermeability of the tumor , but the size of the tumor did not change.9 von buelow reported one single case treated with systemic anti - vegf treatment ( bevacizumab ) but with limited benefit . moreover , the systemic treatment could be associated with severe side effects.10 recent reports of combined therapy with anti - vegf and pdt have shown promising results in these lesions.1113 ziemssen et al reported a case of jrch successfully treated with a single combination of intravitreal bevacizumab and pdt . the patient had marked regression of the hemangioma , an increase in va , regression of the scotoma on vf testing , and macular drying that persisted at the 1 year follow - up visit.11 mennel et al reported a patient with jrch in the right eye that was associated with severe macular and peripapillary leakage.12 the patient was treated with two sessions of pdt ( sparing the part of the hemangioma located within the optic disc ) and five injections of bevacizumab . in this single case , the combination of anti - vegf and pdt appeared to be an effective strategy for the treatment of jrch without side effects.12 by combining anti - vegf with reduced fluence pdt , the outline of the primary angioma can be better delineated and may thus reduce the energy and the treatment area , thereby minimizing the damage to the neurological tissues.13 fong et al reported a patient with jrch had associated macular edema and epiretinal membrane.13 the patient was treated with ivr injection and pdt 1 week before pars plana vitrectomy and epiretinal membrane peeling surgery . the use of this triple combined approach resulted in improvement of va that remained stable at the 1 year follow - up visit.13 recently , some researchers have applied intravitreal anti - vegf therapy alone for the treatment of rch . ach et al reported the outcome of intravitreal bevacizumab for a rch located in the midperiphery.14 a total of nine injections ( administered every 1314 weeks ) of 2.5 mg bevacizumab were given intravitreally in the left eye over a period of 26 months . repeated intravitreal injections of bevacizumab stopped the growth of the present rch and perhaps inhibited the development of new hemangiomas in the treated eye . this is supported by the observation of ongoing growth of hemangiomas in the kidney , brain , and untreated fellow eye.14 dahr et al reported two patients with juxtapapillary or large peripheral angiomas secondary to vhl who were treated with intravitreal injections of pegaptanib ( 3 mg/100 l ) , given every 6 weeks for minimum of six injections per patient . one of these two patients had improvement in va , but no significant change in fluorescein leakage or tumor size was detected in either patient . the follow - up was at 1 year , and some adverse effects ( hypotony and tractional retinal detachment ) were reported.15 in our case , the patient had a progressive jrch with no associated leakage or exudates . we performed a single ivr in order to halt the progression of jrch and prevent all possible complications . ranibizumab likely inhibited vegf in and around the tumor and also suppressed permeability via the blockage of vegf . also , ivr seems to have the advantage of a decreased potential for retinal damage compared with other treatments for jrch . the patient was continuously followed on a routine basis to assess any need for further ivr if any progression of jrch was noted . in conclusion , ivr treatment can be effective in the stabilization of jrch resulting from vhl disease and in the prevention of the exudation of hemangiomas . ivr therapy might therefore be considered as an alternative treatment for progressive jrch , especially in patients with normal va . however , longer follow - up and greater numbers of cases are needed to confirm the effectiveness of ivr for progressive jrchs .

however , it should be considered in differential diagnosis with other umbilical masses because it can cause significant morbidity . physical examination showed an irreducible umbilical tumor , the size of olive , with dubious secretion . a definitive diagnosis of umbilical cord and intestinal hemangioma was established after surgical excision and histologic examination of the umbilical mass . this report highlights the diagnostic challenges of hemangiomas in unusual locations . apart from the rarity of these tumors , few tests are available to guide diagnosis , and surgery and histologic examination are generally required for a definitive diagnosis . finally , it is essential to rule out associated malformations and hemangiomas in other locations . hemangiomas are benign endothelial cell neoplasms that preferentially affect the skin , although they are found in many organs . they occur as isolated lesions or as multiple lesions with extensive cutaneous involvement or diffuse involvement affecting the skin and other organs , as diffuse neonatal hemangiomatosis . hemangiomas may also form part of a syndrome or occur in association with other malformations . several hundred cases of placental hemangioma have already been described , but umbilical cord hemangioma is an extremely rare tumor arising from endothelial cells of the umbilical vessels and the umbilical arteries in particular . in a pubmed search of the literature from 1951 , 44 cases have been reported to date , and the tumor is identified prenatally in over 80% of cases and presents as a fusiform swelling in the cord . this swelling corresponds to an angiomatous nodule surrounded by edema of the wharton jelly with a diameter of between 0.2 and 18 cm . only 14 cases were detected after delivery , 7 of them as part of the post mortem investigation , ( table 1 ) . reported associations include increased alpha - fetoprotein levels , hydramnios , fetal hydrops , and cord hemangiomas are assumed to increase the risk of perinatal mortality and morbidity . the case presented here is of an umbilical cord hemangioma diagnosed in neonatal period , which was mimicking other initially diagnosis . for publishing , informed consent was obtained by the parents and no other ethical accuracies were necessary . a 12-day - old full - term female infant born by vaginal delivery after an uneventful pregnancy was referred to our hospital with a suspected diagnosis of omphalitis and umbilical hernia . it was the first pregnancy of a 30-year - old mother and prenatal ultrasound ( us ) and other examinations had been normal . the infant had an apgar score of 9 and 10 at 1 and 5 minutes , respectively . the mother had no toxic habits ( drugs , alcohol , tobacco , or drugs ) . after the umbilical cord stump had fallen off the next day , we observed a pink , rubbery irreducible umbilical mass measuring 3 2 cm with the umbilical vessels displaced toward the bottom but contained therein . there was a dubious hole and secretion in the center of the mass ( fig . bottom center : urachus . us of the umbilical cord showed a solid hyperechogenic mass , and examination of blood flow with color doppler imaging showed intense flow with interruption in the anterior abdominal wall and an apparent connection to the dome of the urinary bladder . abdominal us showed a hypervascular solid mass with hyperechoic areas on the left flank and medial extension across the midline . the initial question was whether the umbilical and intestinal masses might be related or whether they were 2 independent processes . evaluation of tumor markers for the differential diagnosis of abdominal masses showed unremarkable findings for urinary catecholamines , alpha - fetoprotein , neuron - specific enolase , and human chorionic gonadotropin . abdominal magnetic resonance imaging ( mri ) confirmed the presence of a mesenteric mass in the left flank , with prominent vascularization ; the mass was hyperintense on t2-weighted images and hypointense on t1-weighted images . a solid umbilical mass , with a similar appearance , was observed , but there was no continuity with the bladder dome or the abdominal mass ( fig . the umbilical mass appears to be in contact with the bladder , but it is not continuous with the abdominal component . it was decided to surgically remove the umbilical mass and perform an exploratory laparotomy . the mass showed no intra - abdominal continuity and contained the umbilical vein , 2 umbilical arteries , and the obliterated urachus . the right umbilical artery showed vascular abnormalities consistent with aneurysmatic dilatation and tortuosity . in the abdomen , we detected a vascular malformation in the territory supplied by the superior mesenteric artery , the mesentery extending from the jejunum to the distal ileum and the transverse colon wall were affected ( fig . echocardiography , us examination of the brain , eye examination , and thoracic mri were normal . the patient was treated with oral propranolol at a dosage of 2 mg / kg / day in 3 divided doses , and no adverse effects were observed . us imaging of intestinal hemangioma after 6 months of propranolol treatment showed a decrease in the caliber of vessels and distal vessels are not accentuated . the differential diagnosis includes hematomas , varicose veins , aneurysms , thrombosis , hernia into the cord , abdominal wall defects , omphalomesenteric duct cysts , allantoic cysts , and tumors such as teratoma and metastatic neuroblastoma . diagnosis by direct visualization and imaging studies can be difficult . in our patient , for example , the initial diagnosis contemplated was urachal or omphalomesenteric remnants . associated cord edema is a nearly constant finding in umbilical cord hemangiomas . these tumors typically occur as an isolated anomaly , although they have been described in association with other malformations , such as heart malformations , anencephaly , skin malformations , various syndromes , and , like in our case , cutaneous / systemic hemangiomas . they have been associated with a mortality rate of 35% ; the main causes of death are bleeding and umbilical cord torsion . gastrointestinal hemangiomas are rare and account for just 0.05% of all intestinal neoplasms ; they are more common in the small intestine , and the jejunum is the most frequently affected segment . our case is particularly interesting , as there have been few reports of involvement of the mesentery and the transverse colon , and to the knowledge of the authors , this is the first report showing association of umbilical cord and intestinal hemangiomas detected in the neonatal period . preoperative diagnosis of a mesenteric hemangioma is virtually impossible although abdominal imaging can help to locate and visualize the lesion . treatment varies with the type of lesion , location , extent of involvement , symptoms , and general operability . expectant management only may be indicated in small hemangiomas , located far from areas of possible functional damage and slow - growing . hemangiomas that can cause functional impairment , fast - growing hemangiomas , disseminated cutaneous hemangiomas , and visceral hemangiomas that can lead to heart failure , severe bleeding , or coagulation disorders must be treated . patients with gastrointestinal hemangiomas located in a short well - defined intestinal segment are therefore usually candidates for surgical resection . other treatments for nonresectable or diffuse hemangiomas include radiation therapy , cryotherapy , brachytherapy , sclerotherapy , arterial embolization ( with limited success ) , and pharmacologic treatment . the use of multiple treatments can be explained by our limited knowledge of the biology of hemangiomas . the traditional drugs of choice were corticosteroids and drugs such as thalidomide and somatostatin but propranolol is now the first - line treatment for problematic proliferating hemangiomas . in our patient , we initiated propranolol treatment because of the extent of the lesion and the potential risk of serious complications , and the response was favorable . in summary , although umbilical cord hemangiomas are rare and are usually identified prenatally , clinicians should understand their clinical significance and include these tumors in the differential diagnosis of an umbilical mass in a newborn . a thorough physical examination and complementary studies are necessary to evaluate associated anomalies , including cutaneous and systemic hemangiomas and other malformations . diagnosis of umbilical cord or abdominal masses is challenging and a definitive diagnosis usually requires surgery and histopathologic examination . finally , clinicians should be aware that despite their benign nature , intestinal hemangiomas can lead to a fatal outcome in cases of delayed diagnosis and inappropriate treatment .

trigeminocardiac reflex is defined as the sudden onset of parasympathetic dysrhythmia , sympathetic hypotension , apnea and gastric hyper motility under stimulation of any sensory branches of the trigeminal nerve . sensory terminals of the trigeminal nerve sends nerve signals , due to the extra - ocular muscle stretch or increased intraocular pressure , from the ciliary ganglions of ophthalmic branch of the trigeminal nerve to the sensory nerve cells within forth ventricle , which in the efferent pathway vagus nerve goes to the heart and lead to bradycardia , reduced cardiac conduction and cardiac contractility . this reflex , which is caused by the vagal stimulation is classically named oculocardiac reflex . it occurs through ophthalmic branch of trigemino nerve . although this reflex commonly occurs during the strabismus surgeries , it can possibly happen in other ophthalmological surgeries . previous interventions which have been applied to prevent this reflex in strabismus surgeries were either atropine or xylocaine or both . intravenous ( iv ) atropine blocks peripheral muscarinic receptors in the heart ; moreover , xylocaine hydrochloride ( retro bulbar ) blocks the conduction in the ciliary ganglion in the afferent pathway of the reflex arc . the advantages of topical anesthesia include rapid recovery of vision , cost effectiveness and avoiding complications that might occur following injection by needle . in addition , risk factors for this reflex are hypercapnia , hypoxia , acidosis , sedation and drugs . in the study of ruta 2 percen t xylocaine has been proved to be effective in controlling oculocardiac reflex while applying topically upon rectus muscles in strabismus surgeries . in another study in which topical anesthesia has been used instead of general anesthesia for strabismus surgeries , the prevalence of oculocardiac reflex has been rarely reported . in a study upon patients undergoing retinal and strabismus surgeries , to assess the effects of topical xylocaine compared with placebo , occurrence of this reflex was significantly reduced . in another study , in scleral buckling under general anesthesia , applying local xylocaine in opened conjunctiva 2 percent topical xylocaine is not commonly applied in ophthalmological surgeries under general anesthesia and also most of these patients are old and generally take beta blockers and calcium channel blockers , hence this study carried out in feiz medical center science with the aim of comparing the preventive impact of 2 percent topical xylocaine and iv atropine on oculocardiac reflex in ophthalmological surgeries under general anesthesia . in a double - blind randomized clinical trial study , after obtaining approval from research ethics committee of isfahan university of medical sciences , 150 patients aged of 18 - 90 years , were enrolled in the study . at first , aim of the study was explained to the participants and their written informed consent was obtained . study samples were patients undergoing ophthalmological surgeries such as retinal detachment and vitrectomy with scleral buckling in feiz medical center affiliated to the isfahan university of medical sciences . the inclusion criteria are defined as patients between the ages of 18 - 90 , candidate for retinal detachment surgery and vitrectomy and not allergic to xylocaine and similar compounds . on this basis , patients who had taken any drugs or any substance which have interaction with xylocaine or atropine , patients with cardiac arrest and those who needed any other ophthalmological surgeries except the two above mentioned ones as inclusion criteria in this study . among 150 patients who met the inclusion criteria , 75 patients assigned to each group , by means of a sample size formula to compare two ratios ( occurrence of occulo cardiac reflex in both groups ) , with a confidence level of ( 95 percent ) , power of ( 80 percent ) and the prevalence of ocr that assumed 30 percent in the experimental group based on the literatures and 50 percent in the control group due to uncertainty of the prevalence . patients were randomly allocated into two experimental and control group by means of randomization allocation software ( ras ) randomly assign subjects to different trial groups by computer . in the experimental group patients received xylocaine drop which instilled in desired eye . likewise in the control group received iv atropine . once patients were transferred and positioned on an operating table , monitoring equipment 's such as electrocardiograph , pulse oximetry and blood pressure cuff were connected . induction of anesthesia was done by applying 4 milligram / kg thiopental sodium , 0.035 milligram / kg midazolam , 1.5 microgram / kg fentanyl and 0.6 milligram / kg atracurium . after tracheal intubation , anesthesia continued by a combination of 50 percent of n2o in o2 and 1.5 percent isoflurane . when anesthesia started , four drops of 2 percent xylocaine with an interval of 2 minute during 10 minute was instilled into the desired eye of the patients in the experimental group . in the control group , tidal volume and respiratory rate of samples were adjusted 10 milliliter / kg and 12 times / minute , respectively . afterwards , during the operation , by monitoring of end - exhaled carbon dioxide was monitored and maintained 25 - 35 by adjusting respiratory rate and tidal volume . furthermore , heart monitoring and pulse oximetery was performed to evaluate and record heart rate , arterial oxygen saturation , systolic blood pressure , diastolic blood pressure and mean arterial blood pressure . arterial oxygen saturation was maintained over 95 percent ; in addition , in case of reducing the heart rate lesser than 20 percent of baseline , it was considered as bradycardia and recorded in the questionnaire . at the end of the operation , memory of monitoring equipment was checked and the minimum and maximum heart rate were recorded in the questionnaire by one of the researcher . for the purpose of double- blinding in this study , neither the technician who recorded the memory nor the patients were aware of the administered treatment and patient group . in case of reducing heart rate to less than 60 beats if needed , it was repeated after 3 minute . in case of dropping systolic blood pressure to lesser than 30 percent of baseline , it was recorded as hypotension and in case of dropping the systolic blood pressure to less than 90 mmhg , it was treated by reducing the anesthetic drugs and extra fluid therapy . in the case that did not response to the treatment , 5 mg of ephedrine was injected and also repeated as needed . at the end of anesthesia , the residual effect of muscle relaxants was reversed by applying 0.02 milligram / killogram of atropine and 0.05 milligram / kilogram of neostigmine . data were collected , edited and analyzed by statistical package for the social sciences software version 20 . statistical tests used to analyze data are chi - square test ( for comparison of frequency distribution of qualitative variables between two groups ) and repeated measures anova for comparison of hemodynamic variables during study . in this study , the amounts of p < 0.05 were considered to be significant . a total of 154 patients were recruited to the study and four of them were excluded [ figure 1 ] . in table 1 , distribution of demographic characteristics and underlying variables has been shown for samples of two groups . according to the table , no significant differences were observed between two groups regarding the distribution of these variables . in table 2 , anova with repeated observations showed that the mean of changes in heart rate of samples who received xylocaine and atropine is significantly different = 0.003 ) . the mean of minimum heart rate was ( 60.1 9.2 ) in xylocaine group and ( 71.2 13.5 ) in atropine group . according to the t - test , in addition , the mean of maximum heart rate in both experimental and control groups were ( 88.9 16.8 ) and ( 97 16.5 ) respectively . t - test indicated that the difference between these two groups is significant ( p = 0.002 ) . in table 3 , the frequency distribution of complications in two groups who received xylocaine and atropine has been indicated . in this regard , the incidence of bradycardia , dropping heart rate to less than 60 beat / minute , hypotension and dropping the blood pressure to the less than 90 mmhg in the xylocaine group was significantly more than that of atropine group ; moreover , non - sinusoidal rhythm and cardiac arrest did not happen to any patients of the two groups . for six patients who received xylocaine , furthermore , six patients in xylocaine group ( 8 percent ) and seven patients in atropine group ( 9.3 percent ) received ephedrine . on this basis , chi monitoring the hemodynamic variables of patients throughout the intervention showed that systolic , diastolic and mean arterial blood pressure did not have any significant differences between two groups during 120 min . however , heart rate changes had significant differences between two groups , so that the heart rate was higher in patients receiving iv atropine . as it was already explained , atropine is an anti - cholinergic drug which prevents bradycardia . in our study , the incidence of bradycardia in patients who received topical xylocaine was 90.7 percent and in patients who received atropine was 17.3 percent . both groups showed a significant difference in this regard . in 40 percent of the patients who received xylocaine , heart rate dropped to less than 60 beats / min , whereas it only happened in 13.3 percent of patients who received atropine . the incidence of hypotension in patients who received atropine ( 68 percent ) was significantly less than that of the patients who received xylocaine ( 24 percent ) . particularly , severe dropping of blood pressure ( dropping the blood pressure less than 90 mmhg ) , which can be problematic during anesthesia was lesser in the atropine group in compare with xylocaine ( 28 percent vs. 8 percent ) . consort diagram showing the flow of participants through each stage of the study distribution of demographic variables in two groups hemodynamic indexes for the two groups the frequency distribution of complications in xylocaine and atropine groups in this study , a total of 150 patients were allocated into 2 groups applying xylocaine and atropine . two groups did not have any significant differences in respect of demographic and basic variables including age , sex , type of operation , duration of operation , taking beta blockers , taking calcium - blockers , history of high blood pressure and taking anti - hypertensive drugs . hence , the confounding effect of these factors has been neutralized and differences are likely related to the type of medication that these patients take . monitoring the hemodynamic variables of patients during the intervention showed that systolic , diastolic and mean arterial blood pressure did not have any significant differences between two groups from the onset to the min 120 . however , heart rate changes had significant differences between two groups , so that the heart rate was higher in patients receiving iv atropine . as it was already explained , atropine is an anti - cholinergic drug which prevents bradycardia . in our study , the incidence of bradycardia in patients who received topical xylocaine was 90.7 percent and in patients who received atropine was 17.3 percent . both groups showed a significant difference in this regard . in 40 percent of the patients who received xylocaine , heart rate dropped to less than 60 beats / minute , whereas , it only happened in 13.3percent of patients who received atropine . the incidence of hypotension in patients who received atropine ( 68 percent ) was significantly less than that of patients who received xylocaine ( 24 percent ) . particularly , severe dropping of blood pressure ( dropping the blood pressure less than 90 mmhg ) , which can be problematic during anesthesia was less in the group which received atropine ( 28percent in comparison with 8 percent ) . in a study done by gilani et al . in 2005 , 60 patients undergoing strabismus surgeries under general anesthesia were investigated in two groups for the aim of comparing the effects of iv atropine before inducting anesthesia with placebo , similar results were obtained . in addition , in the placebo group who had received no premedication , dropping the mean of heart rate , incidence of junctional rhythm and the number of patients who needed atropine injection during the surgery were higher and the prevalence of this reflex was 70 percent in comparison with the experimental group which was 10 percent . therefore , the mentioned study also showed that premedication by atropine in patients undergoing strabismus surgery prevents the occurrence of the reflex . in another study done by ruta et al . upon patients undergoing strabismus or retinal surgery , the prevalence of the reflex in the placebo and topical xylocaine groups were 86.1percent and 37.1percent respectively . in addition , repeated episodes of severe bradycardia significantly reduced from 40 percent in the placebo group to 2.9 percent in xylocaine group . however , unlike our study that xylocaine was compared with atropine injection , in this study , xylocaine has been compared with placebo . in a study done by misurya et al . upon patients at the aged of 10 - 30 years undergoing strabismus surgery under general anesthesia a group of patients received 0.6 mg of atropine sulfate before anesthesia and finally the reflex was seen just in 10 percent of patients , which did not have any significant difference with the control group . another group of patients received 1 cc of 2 percent xylocaine for retro bulbar block and the reflex was seen in 20 percent of them , which did not show any significant differences with the control group . the third group of patients received both retrobulbar xylocaine and premedication of atropine , finally the reflex was seen in none of them and they had a significant difference with the control group . in fact , in the study , it was concluded that using a combination of both drugs in comparison with using each drug separately is more effective to prevent the reflex in strabismus surgeries . in another study done by dehghani et al . in feiz medical center of isfahan upon the patients undergoing scleral buckling surgery due to rhegmatogenous retinal detachment , the effects of topical xylocaine was compared with the placebo . using xylocaine during the operation for the experimental group significantly reduced prevalence and severity of the reflex . in a study done by snir et al . upon the patients undergoing strabismus surgery under general anesthesia to investigate supplementary effects of topical anesthesia , sub - tenon anesthesia was done by injecting 3.4 cc of a combination of 2 percent xylocaine and 5 percent bupivacaine . according to the results , the incidence of episodes of reflex , arrhythmia and bradycardia was less than that of the control group . although , the prevalence of bradycardia ( dropping heart rate to more than 20 percent of basic ) and blood pressure drop ( more than 30 percent ) in xylocaine group was higher than that of atropine group , the percentage of patients who needed medical intervention ( six patients who were in xylocaine group , also received atropine to treat bradycardia . likewise , six patients in xylocaine group and seven patients in atropine group also received ephederine ) showed no statistically significant differences between these two groups . although most of the ophthalmological patients have the history of cardiac diseases and hypertension and they might take medications for this purpose , but premedication by atropine showed no side - effects and it can be recommended . on the other hand , using topical xylocaine have largely reduced vagus reflex ( bradycardia and hypotension ) . therefore , due to this fact that topical xylocaine does not harm patients , applying this substance in ophthalmological surgeries prior to the onset of general anesthesia can be recommended . it should be noted that atropine and ephedrine can be used therapeutically , if it 's needed . for the purpose of ethical considerations and study limitations , it should be noted that in this study usual drugs , which are normally applied in ophthalmological surgeries have been used for patients ; therefore , no ethical barrier or limitation has been determined . according to this study , though we can recommend both topical xylocaine and iv atropine for prevention of oculocardiac reflex before beginning of surgery during general anesthesia , but the preventive impact of topical xylocaine on this reflex in ophthalmological surgeries such as retinal detachment and vitrectomy with scleral buckling has been less than the effect of iv atropine . because iv atropine had no any side - effects in high risk patients in this study , therefore , iv atropine can be recommended for these patients who suffer from heart problems and hypertension with the history of taking anti - hypertensive drugs .

peripheral arterial disease ( pad ) commonly results from progressive narrowing of arteries in the lower extremities due to atherosclerosis . it has become evident that pad is an important predictor of substantial coronary and cerebral vascular risk [ 2 , 3 ] . the most common symptomatic manifestation of mild to moderate atherosclerotic pad is intermittent claudication , observed in ~5% of individuals older than 60 years [ 5 , 6 ] . several studies , using the ankle - brachial index , have revealed that the presence of pad , even when being asymptomatic and in patients with no history of other cardiovascular diseases , is a marker of greatly increased cardiovascular morbidity and mortality [ 7 , 8 ] . oxidative stress , through the extra generation of reactive oxygen species ( ros ) , is also involved in the development and progression of pad . moreover , risk factors associated with pad , similar to those for coronary heart disease , include high blood concentrations of total cholesterol and ldl - cholesterol . inhibitors of 3-hydroxy-3-methylglutaryl coenzyme a ( hmg - coa ) reductase , called statins , are well - known lipid - lowering drugs . statins not only reduce lipid levels , but they also have important pleiotropic effects , improving endothelial function , reducing oxidative stress , affecting the lipid metabolism enzyme activities , and so forth [ 1214 ] . three main reasons have been suggested for initiating statin therapy for patients with pad of the lower limbs : ( 1 ) prevention of coronary heart disease ; ( 2 ) treatment of lower extremity peripheral arterial disease ; and ( 3 ) perioperative treatment of patients with peripheral arterial disease . although several trials have shown that treatment with statins is associated with a reduction in coronary heart disease events , such as stroke [ 18 , 19 ] and mortality [ 2023 ] , most of these trials did not specifically address the treatment of patients with pad . the aim of the present study , therefore , was to evaluate lipid peroxidation parameters and total antioxidant capacity in the serum of patients with pad before surgery as well as 35 days and 710 days after surgery . we also compared these parameters with those in a group of patients receiving simvastatin therapy . the study group consisted of pad patients , treated either surgically ( group i , n = 35 males , aged 61 8 years ) or pharmacologically ( group ii , n = 18 males , aged 62 7 years ) in the department of general and vascular surgery at the poznan university of medical sciences . patients included in group i were admitted for surgical treatment ( implantation of an aortobifemoral bypass graft ) . they were diagnosed by measurement of the ankle brachial index ( abi ) and arteriography of the lower limbs , and were clinically stable without any accompanying diseases . the patients treated surgically had shown critical ischemia ( abi less than 0.90 , rest pain , ulcer or necrosis of the lower limbs , and ankle pressure 50 mmhg ) . blood samples were collected without anticoagulant , in the fasting state , before surgery as well as 35 days and 710 days after surgical treatment . group ii had been receiving statin therapy , with 20 mg of simvastatin daily , for at least 3 months . the study protocol was approved by the ethics committee of poznan university of medical sciences , and the subjects were fully informed and gave their written consent . all the reagents were of analytical grade of purity ( sigma - aldrich , st . spectrophotometric measurements were carried out on sp-8001 uv / visible spectrophotometer ( metertech inc . , taiwan ) . the subjects ' lipid profile ( total cholesterol , hdl cholesterol , ldl cholesterol , and triglycerides ) , glucose , uric acid , apolipoproteins ( apo a - i , apo b ) and fibrinogen concentrations in serum were measured by standard laboratory procedures , using the roche cobas 6000 analyzer system , dade behring bn ii system , and the sysmex ca-500 series system . the total antioxidant potential ( capacity ) of serum was determined by ferric reducing antioxidant power ( frap ) assay according to the benzie and strain method , based on the reduction of ferric ion fe(iii ) to ferrous ion fe(ii ) at a low ph . this method measures the change in absorbance at 593 nm owing to the formation of a blue - colored fe(ii)-tripyridyltriazine complex from the colorless oxidized fe(iii ) form , by the action of electron - donating antioxidants . lipid peroxidation was measured by estimation of both lipid hydroperoxides ( loohs ) and malondialdehyde ( mda ) concentrations by the sodergren method and ohkawa et al . the concentration of thiol ( -sh ) groups was determined according to the hu method . ceruloplasmin , in the presence of o - dianisidine hydrochloride in an acidic solution ( ph 5.0 ) , forms a stable yellow - brownish product . absorbance was measured at 540 nm and ceruloplasmin activity was expressed in u / l ( mol of oxidized substrate in 1 ml of serum in 1 min ) . the paired student 's t - test ( to compare the values before and after surgery ) , the two - tailed unpaired t - test ( to compare group i and group ii ) , and the f - test were used for statistical analysis of the data . the mean changes ( ) in lipid peroxidation parameters and antioxidant capacity , that is , 35 days ( 1 ) and 710 days ( 2 ) after surgery , in relation to the values obtained before surgery , were calculated . pearson 's correlation coefficients r1 and r2 were computed for the results found before surgery and the changes observed during treatment ( 1 , 2 ) . as shown in table 1 , generally , the clinical parameters of the two groups of patients included in this study did not differ significantly . however , the concentrations of total cholesterol and ldl cholesterol were found to be significantly higher in group i before surgery than those in group ii on simvastatin therapy . group i patients before surgery had enhanced oxidative stress , as demonstrated by elevated serum levels of mda , in comparison to those treated pharmacologically . we did not observe any significant decrease in looh and mda concentrations in group i patients after surgery ( figures 1 and 2 ) . the total antioxidant capacity ( expressed as frap ) and -sh group concentrations in the serum of patients on statin therapy and in those before surgery were similar ( figures 3 and 4 ) . however , a decrease was observed in frap level 35 days after surgery , and 710 days after surgery , it was significantly lower in comparison to the values before surgery and on simvastatin therapy . moreover , thiol group concentrations 35 days postoperatively were lower than those before surgery and 710 days after surgery and were lower than those in patients treated with simvastatin ( figure 4 ) . a significant difference in the oxidase activity of ceruloplasmin between group ii and group i before surgery was observed . its oxidase activity in serum was higher in group ii and 710 days after surgery in group i , in comparison to the values before and 35 days after surgery ( figure 5 ) . in addition , all the studied parameters ( looh , mda , frap , -sh groups , and ceruloplasmin ) in group i patients before surgery were significantly correlated with mean changes at 35 days ( 1 ) and/or 710 days ( 2 ) after surgery ( table 2 ) . oxidized ldl is a biochemical marker of oxidative damage and has atherogenic effects , including the stimulation of foam cell formation and activation of the inflammatory process in the vascular wall . the findings of the present study are broadly consistent with the hypothesis that lipid peroxidation may be one mechanism through which several risk factors may promote pad . , we observed an increased mda level in the patients before surgery , which suggests that they were in a state of high oxidative stress . at neutral ph , mda exists as a low reactive enolate anion , but it is still toxic and may damage many biologically important molecules . determination of the lipid peroxidation product mda in pad is important because , as was shown earlier , mda damages collagen by forming transverse intramolecular bonds that cause rigidity of blood vessels . in addition , modification of native ldl by mda causes the accumulation of cholesteryl esters within the cell in which the atherosclerotic reaction takes place . several trials have shown that statin therapy relieves symptoms and improves function in patients with lower extremity pad [ 32 , 33 ] . mda concentrations in the serum of the patients treated with simvastatin were lower than those in group i patients before surgery . since pad patients are usually undertreated with regard to the use of lipid - lowering agents , compared to patients with coronary heart disease , our results provide strong support for the efficacy of statin therapy in pad . human cells and tissues contain many antioxidant enzyme systems and nonenzymatic antioxidants , which protect against the action of toxic free radicals . the frap assay used here is a useful indicator of the body 's antioxidant status , which determines the level of non - enzymatic antioxidants , such as uric acid , ascorbic acid , -tocopherol , and some protein - containing -sh groups . an increase in frap level in the patients before surgical treatment is consistent with similar results obtained by gawron et al . in a group of patients with coronary heart disease and by lantos et al . in patients with hypertension . an increased antioxidant capacity of serum seems to be response to an increase in the concentration of lipid peroxidation products . the reduction in frap level observed in our group i patients 35 and 710 days after surgery can be partly explained by a decrease in their serum antioxidant status and an increase in serum oxidant levels following surgery , as indicated by the negative correlations found ( table 2 ) . in their study , girona et al . they showed that simvastatin therapy increases the resistance of both ldl and hdl to oxidation . in addition , simvastatin inhibits the ability of activated macrophages to oxidize ldl in vitro in a dose - dependent manner . a similar effect has been shown for other statins , such as atorvastatin , which reduces ldl levels and hdl oxidation and protects paraoxonase an enzyme bound to hdl . with regard to frap in our study , simvastatin therapy ( 20 mg daily ) did not influence the total antioxidant potential . however , shin et al . found a significant increase in antioxidant ability in their group of hypercholesterolemic patients treated with 40 mg simvastatin daily . they suggested the possibility of a dose - dependent effect of simvastatin on plasma antioxidant status . this could be the reason why we did not detect any effect of simvastatin on frap in our study . glutathione and other proteins , such as albumin , are an important part of serum antioxidant activity against free radicals . the thiol groups react with peroxide radicals in the first stage of the oxidation reaction and also act in the lag phase , which is an accepted marker of serum oxidizability . in our group of patients with pad , the decrease in thiol group concentrations , observed 35 days after surgery , followed by an increase 710 days after surgery , may be explained by oxidative stress intensification in reperfusion and by the actions of -sh groups , leading to their reduction . furthermore , a decrease in both lag phase and albumin level has also been reported in the serum of patients with aneurysmal or arterial occlusive disease . some studies have confirmed that ceruloplasmin is an independent risk factor for cardiovascular disease [ 43 , 44 ] . authors of that study suggest that increased ceruloplasmin activity is related to the acute phase response to inflammation and necrosis in atherosclerosis obliterans and results from increased ros production , mostly of superoxide radicals . therefore , the preferential oxidative destruction of thiol groups can explain the greater susceptibility of ceruloplasmin to oxidative stress . recent data show that statins may reduce oxidative stress by decreasing the generation of ros , thereby synergizing with the biological effects of antioxidants . the results of our study show that ceruloplasmin activity was influenced by both simvastatin therapy and surgical treatment , but only 710 days postoperatively . in our opinion , simvastatin may be implicated in antioxidant properties of ceruloplasmin , as reflected by the increase in its activity in men on simvastatin therapy . several in vitro studies have indicated that ceruloplasmin is a potent catalyst of ldl oxidation and that has been attributed to the presence of a reduced copper atom on its surface . ros dissociate copper from ceruloplasmin and therefore can markedly alter its structure and function , which could explain the initial lack of differences in its activity 35 days after surgery . in view of the escalating prevalence of pad in our aging population , further studies are needed to clarify precisely not only the mechanisms involved but the relative importance of the lipid - related and nonlipid - related effects of statins as well . results of this study show diverse oxidative state responses to surgical treatment and confirm the beneficial effects of statin therapy in pad .

the process of optimization of dose distribution is well recognized as one of the most important parts of the whole treatment planning procedure in modern brachytherapy . thus the implemented optimization routines are essential modules of almost all clinically used treatment planning systems . especially when high dose rate brachytherapy is used , minimal alteration of a single dwell time could result in the need for major modification of the whole treatment plan . nowadays it is almost impossible to prepare an acceptable treatment plan in a reasonable time by entering dwell times manually . in order to achieve the best treatment outcome optimization algorithms when the catheter reconstruction is performed very precisely using 3d imaging methods including us , ct and mri it is essential to preserve high quality of the whole procedure by using trusted dose calculation and optimization algorithms [ 13 ] . treatment planning system commissioning is one of the most important parts of the quality assurance system in a working brachytherapy department . migration to a more sophisticated system is always a step forward for the planning team but careful verification of the workflow and obtained results is mandatory . the question is not only whether the quality and safety of the previous standards can be preserved , but also about the possibility of reaching a higher level . the general objective of this study was to compare and verify calculation algorithms implemented in the treatment planning systems plato brachytherapy v.14.3.7 and oncentra masterplan ( brachy planning ) v.3.1 sp 3 . the discussed procedure was a part of commissioning of the brachy planning module before migration from the plato system . in order to compare calculation and optimization algorithms implemented in oncentra masterplan ( omp ) and plato brachytherapy ( plato ) treatment plans for 20 multiplane interstitial hdr breast implantations were prepared [ 5 , 6 ] ( fig . 1 ) . reconstructed geometry of the application and sample dose distribution for multiplane hdr breast implant for reconstruction of the catheter geometry and definition of the tumour bed area , ct images were used with 3 mm slice thickness . to avoid inconsistencies concerning delineation of the target , the clinical target volume ( ctv ) was reconstructed only once in the target definition module of oncentra masterplan and exported as a dicom rt file to the plato system . before the export procedure the target structure was simplified and the number of vertices was reduced to 50 to avoid errors in the plato import module ( the older system does not accept contours that are too detailed 99 points is the high limit ) . to ensure that there were no differences concerning the ctv volume student s t - test for related variables ( = 0.05 ) implant geometry ( catheters ) was reconstructed by one physicist using both treatment planning systems to minimize the observer - related catheter reconstruction deviation . after reconstruction of the catheter geometry , only dwell positions inside the ctv were activated . for the oncentra masterplan the dose was prescribed at the 50 dose points located at the surface of the ctv . for the plato system points optimization at dose points with the distant option were used in both planning systems , as this optimization technique was most commonly used in the greater poland cancer centre brachytherapy department [ 911 ] . for verification and comparison of obtained dose distributions the authors decided to use commonly known parameters : d90 , d100 , v100 , v150 , v200 and dnr ( dose non - uniformity ratio ) . only the ctv volumes were used as absolute values ( ccm ) [ 7 , 12 ] . d90 and d100 were calculated as the dose deposited in 90 and 100 percent of the ctv volume respectively . v100 , v150 and v200 were calculated as the percentage fraction of ctv where the deposited dose was 100 , 150 and 200 percent of the prescribed dose respectively . all the parameters were compared using student s t - test for related variables ( = 0.05 ) . no manual correction of the dose distribution was performed as the main aim of the study was to compare parameters obtained using implemented algorithms [ 911 ] . mean values of ctv volume calculated in plato and omp were 49.72 ccm and sd was 21.79 for both treatment planning systems . the assumption based on results of statistical analysis is that there were no statistically significant differences between ctv volume calculated using plato and omp . no statistically significant differences ( p = 0.68 ) concerning volume receiving 100% of the prescribed dose was noted . mean value of v100 was 76.06 7.42% for plato and 76.60 5.67% for omp . for the high dose area parameters mean values of v150 were 31.17 9.46% for plato , and 28.87 8.54% for omp . the observed v200 values were 12.67 3.28% for plato and 11.97 2.91% for omp respectively . . calculated volume of the ctv ( ctv vol ) and v100 , v150 , v200 parameters and results of the data analysis are presented in table 1 and fig . volume of the ctv and dose distribution parameters for both used treatment planning systems mean values and standard deviations for v100 , v150 and v200 for both treatment planning systems statistical differences between the d90 parameter calculated in both planning systems were not significant either 0.41 0.10 for plato and 0.37 0.10 for omp ( p = 0.02 ) . obtained values of d90 , d100 and dnr with results of statistical analysis are presented in table 2 and fig . 3 . calculated values of d90 , d100 and dnr with results of statistical analysis , for both treatment planning systems mean values and standard deviations for d90 , d100 and dnr for both treatment planning systems no observed differences among ctv volume , v100 , v150 , v200 and d90 suggest that plato brachytherapy and oncentra masterplan could be used interchangeably in clinical practice . on the other hand , this study shows that the dose rate is more homogeneous for plans prepared with omp ( p = 0.02 ) . that suggests that there should be differences among v100 and v150 ( dnr = v150/v100 ) . a closer look reveals that the difference in v150 between plans prepared with omp and plato is close to statistical significance ( p = 0.09 ) . furthermore , the dose distributed to the whole ctv volume is higher in plans prepared using omp . probably it is also possible to achieve an identical dwell position / time pattern for both treatment planning systems , but the aim of this study was to compare and verify the obtained results and to check whether the dose distributions are clinically acceptable . dose distributions for the multiplane hdr breast implants , calculated using optimization algorithms implemented in oncentra masterplan ( brachy planning ) , are faultless from the physicist s point of view . obtained results are also clinically acceptable and most of the parameters do not differ significantly from those calculated using the trusted plato system .

frantz described first time in 1959 about a rare and characteristic tumor arising from body and tail of the pancreas known as solid pseudo papillary tumor ( spt ) of pancreas . it usually affects young women , with a 10:1 predominance over men , at an average age of 24 years . spt usually present with an abdominal mass , abdominal pain or discomfort , but sometimes , it is an incidental finding on imaging studies for other reasons . many of the times , general surgeons are unaware of the spt until the diagnosis is made by computed tomography ( ct ) scan of the abdomen , especially when it is presenting as acute abdomen . we report a case where a female child with spt presented as acute abdomen with hemoperitoneum . in few case reports , the causes of rupture being blunt trauma to the abdomen , and while spontaneous rupture is quite uncommon . a female child aged 12 years , initially presented to a surgeon elsewhere , with a history of sudden onset of severe pain in the upper abdomen and no history of trauma to the abdomen . the surgeon on abdominal examination found to have a vague epigastric mass associated with tenderness . then the patient underwent ultrasonography of the abdomen and suspected to have the possibility of internal bleeding within pseudocyst of the pancreas , occupying whole of the lesser sac . there was severe pain in the abdomen with tachycardia and bp was 100/60 mm of hg . as other routine blood investigations were within normal limits , patient was subjected to ct scan of the abdomen , which revealed a well - demarcated mass of size 10 cm 13 cm arising from the body of pancreas , which was composed of a solid - cystic portion with enhancement of contrast in solid portions and occupying the whole of the lesser sac , posteriorly abutting to the splenic vessels with a breach in the anterolateral part of the capsule ( rupture ) of tumor with minimal hemoperitoneum . the impression was spt arising from the body of pancreas without any metastasis to the liver . , there was a significantly large tumor mass arising from the body of the pancreas , extending to the left subhepatic region , displacing the gastrohepatic ligament anteriorly . there was minimal hemoperitoneum , which may be due to rupture of the tumor . with meticulous dissection whole mass the dissection to separate splenic vessels from tumor and tail of pancreas was possible ; in view of low malignant potential of tumor and without any metastasis , it was decided to preserve the spleen . the head of the pancreas was preserved by dividing the head from the body by using staplers . the histopathological examination ( hpe ) of specimen revealed an encapsulated tumor with hemorrhagic and necrotic areas admixed with solid areas . this case of spt demonstrates how rupture of tumor capsule and hemorrhage within the tumor as reported in hpe , can cause hemoperitoneum and severe abdominal pain , respectively , presenting as acute abdomen . the causes of rupture are , either trauma to abdomen or spontaneous , due to hemorrhage within tumor . spt of pancreas usually not diagnosed on clinical examination , unless a ct abdomen is done to reveal it . even radiologically the differential diagnoses for spt are , solid and cystic lesions of pancreas such as serous microcystic adenoma , cystadenocarcinoma , mucinous cystic neoplasms , other congenital and acquired postinflammatory and infectious cysts . the spt is slow - growing and less aggressive than that of many other pancreatic tumors , and its prognosis is better . surgical removal of the tumor will result in almost total survival ( > 95% ) for those patients with tumors confined to the pancreas and no metastasis . it has a low potential for local infiltration thus recurrence is rare following complete excision . the metastasis occurs only in up to 15% of cases , usually synchronous and confined to the liver or peritoneum . distal pancreatectomy combined with or without splenectomy can be performed for pancreatic body and/or tail tumor , and pancreatoduodenectomy for pancreatic head tumor . the role of chemotherapy and radiotherapy remains to be studied . at present , the surgical procedure of choice for spt still remains controversial . if possible the surgical procedures are preserving pancreatic parenchyma and function should be selected , considering the nature of spt . similarly in our case , local excision of the tumor with preservation of head of pancreas and spleen has been done . in recent years , laparoscopic procedures of enucleation and distal pancreatectomy have already been done and have shown that this approach can be considered for the low - grade malignant spt . it was mentioned that there were no statistically significant differences in the postoperative complications and the prognosis between laparoscopic surgery and open surgery for spt . in acute cases , like our case with rupture of spt , recently , the robotic distal pancreatectomy is becoming as a possible minimally invasive technique for patients with spt . a relatively uncommon case of hemorrhage within tumor and spontaneous rupture of spt of the pancreas , presenting as acute abdomen in a female child has been described . we were able to locally resect spt completely with preservation of head of pancreas and spleen .

due to the importance of electrostatic interactions in biomolecular systems , a variety of computational methods have been developed for evaluating electrostatic forces and energies [ see ( 16 ) and references therein ] . typical computational electrostatics methods for biomolecular systems can be loosely grouped into two categories : explicit solvent methods , which treat solvent molecules in full molecular detail , and implicit solvent methods , which include solvent solute interactions in averaged or continuum fashion . implicit solvent methods are , by definition , limited in detail and therefore lack the atomic - scale accuracy of their explicit solvent counterparts . however , implicit solvent methods have gained increasing popularity , in part due to their elimination of the extensive sampling of solvent configurations required with explicit models ( 1,37 ) . the basic ingredients of an implicit solvent electrostatics calculation are environmental parameters such as temperature , solvent dielectric and ionic strength ; biomolecular atomic coordinates ; and parameters for atomic charges and radii . while the environmental parameters are relatively straightforward to specify , the remaining two ingredients can often be difficult to supply . in particular , most biomolecular structures in the protein data bank ( pdb ) ( 8) do not contain hydrogen atoms , and many are also missing a fraction of the heavy atom coordinates . the addition of hydrogens and the reconstruction of these missing coordinates is not a trivial process ; electrostatic properties obtained from the repaired structures can often be very sensitive to the manner in which missing atoms are added and protonation states are assigned ( 9,10 ) . furthermore , inconsistent atomic nomenclature and other force field idiosyncrasies can often make the assignment of atomic charges and radii a cumbersome task . an additional obstacle to the use of pdb structures in electrostatics calculations and other biomolecular computational tasks is the accurate assignment of parameters to non - standard residues and ligands . previously ( 9 ) , we introduced the freely available pdb2pqr service ( http://pdb2pqr.sf.net/ ) , which was designed to facilitate the setup and execution of continuum electrostatics calculations from pdb data , particularly by non - experts . the original pdb2pqr server automated many of the common tasks of preparing structures for continuum electrostatics calculations , including adding a limited number of missing heavy atoms to biomolecular structures , estimating titration states and protonating biomolecules in a manner consistent with favorable hydrogen bonding , assigning charge and radius parameters from a variety of force fields , and finally generating pqr output ( a pdb - like format with the occupancy and temperature factor columns replaced with charge q and radius r , respectively ) compatible with several popular computational biology electrostatics [ apbs ( 10 ) and mead ( 11 ) ] , docking [ autodock ( 12 ) ] , simulation [ amber ( 13 ) ] and visualization [ vmd ( 14 ) , pymol ( 15 ) and pmv ( 16 ) ] packages . since its inception , we have continued to expand the capabilities of the pdb2pqr server to address the challenges associated with ligand parameterization in pdb files and to include several new features . the pdb2pqr web service is driven by a modular , python - based collection of routines , which provides considerable flexibility to the software and permits non - interactive , high - throughput usage . the service is available via a number of web mirrors listed at http://pdb2pqr.sf.net/. the source code is also available for download from this link , and due to the portability of python , pdb2pqr can be executed on a wide range of platforms . figure 1 outlines the typical workflow of a pdb2pqr job and summarizes the features described in more detail below . the procedures for reconstruction of missing atoms , hydrogen optimization and apbs input generation were described previously ( 9 ) and are essentially unchanged in the current version of the software . since their initial development , these atom reconstruction options have been greatly improved through a number of bug fixes and code optimization , robust support for separate biomolecular chains , and improved chain termini optimization . the process begins with an input pdb file and ends with a parameterized pqr file and , optionally , an apbs input file . the process begins with an input pdb file and ends with a parameterized pqr file and , optionally , an apbs input file . protonation states for titratable protein groups are assigned by propka 1.0 ( http://propka.ki.ku.dk ) ( 17 ) . propka utilizes a very fast empirical method to predict pka values and is successful at predicting unusual pka values . recently , a comparative study of several protein pka prediction methods showed that propka was the most accurate method overall ( 18 ) . propka uses a heuristic method to compute the pka perturbations due to desolvation , hydrogen bonding and charge charge interactions . in the current version of propka , contributions from nucleic acids as well as heteroatoms such as bound ions or ligands to the pka values are not included . note that , during the course of titration state assignment , propka generates statistics on residue hydrogen bonding , location and solvent accessibility and coulombic interactions . this information is available to users as a downloadable text file provided at the end of the pdb2pqr / propka calculation . pdb2pqr currently allows users to assign protein and ( where available ) nucleic acid parameters based on explicit solvent amber99 ( 19 ) and charmm27 ( 20 ) force fields , the parse continuum electrostatics force field ( 21 ) , a poisson boltzmann - optimized force field by tan et al . user - defined parameters can be uploaded to the pdb2pqr server in a simple flat - file format described in the pdb2pqr user guide . additionally , pdb2pqr output can be customized to include a variety of atom naming schemes , including amber99 ( 19 ) , charmm22 ( 20 ) , parse ( 21 ) and an internal naming scheme based on the iupac naming recommendations ( 23 ) . this flexibility in nomenclature was included to facilitate import of pdb2pqr output into other modeling packages . map which is output at the end of every pdb2pqr calculation and presents a table of atoms name / number , residue name , chain name , amber atom type and charmm atom type to aid in the interpretation of parameter assignment and the development of user - defined charges and radii . the calculation of ligand charges necessitates detailed information on molecular structure and protonation states due to the large variation in the covalent structures of small - molecule protein ligands . the current version of pdb2pqr therefore requires the ligand structure , protonation state and formal charge to be specified by the user in the popular mol2 ( 24 ) format . ligand structures in mol2 format are readily available from popular molecular modeling software and free web services such as prodrg ( 25 ) . future versions of pdb2pqr will include a pdb2mol2 parser and automatic assignment of default ligand protonation states from a small - molecule pka database . the calculation of ligand charges in pdb2pqr is based on the partial equalization of orbital electronegativities ( peoe ) procedure developed by gasteiger and marsili ( 26 ) . in the peoe procedure , orbital electronegativities are linked to partial atomic charges q by a polynomial expansion ( = a + bq + cq + dq ) . the coefficients a , b , c and d were optimized by gasteiger and marsili using gas phase data on ionization potentials and electron affinities . we utilize a peoe algorithm , which has been optimized by czodrowski et al . to obtain better agreement between theoretical and experimental solvation energies for a set of small molecules including the polar amino acids ( 27 ) . the resulting peoe_pb charges have been tested for small - molecule complexes with trypsin , thrombin ( 28 ) and hiv protease ( 29 ) , and have been found to give results that are in agreement with experimental values . the current version of pdb2pqr supports an extension directory for user - defined processing of pdb2pqr output . such extensions might include alternative naming schemes , identification and parameterization of other molecule types , additional hydrogen bond processing , etc . however , it is straightforward for users to download the pdb2pqr software and setup their own web servers with additional functionality based on custom extensions . protonation states for titratable protein groups are assigned by propka 1.0 ( http://propka.ki.ku.dk ) ( 17 ) . propka utilizes a very fast empirical method to predict pka values and is successful at predicting unusual pka values . recently , a comparative study of several protein pka prediction methods showed that propka was the most accurate method overall ( 18 ) . propka uses a heuristic method to compute the pka perturbations due to desolvation , hydrogen bonding and charge contributions from nucleic acids as well as heteroatoms such as bound ions or ligands to the pka values are not included . note that , during the course of titration state assignment , propka generates statistics on residue hydrogen bonding , location and solvent accessibility and coulombic interactions . this information is available to users as a downloadable text file provided at the end of the pdb2pqr / propka calculation . pdb2pqr currently allows users to assign protein and ( where available ) nucleic acid parameters based on explicit solvent amber99 ( 19 ) and charmm27 ( 20 ) force fields , the parse continuum electrostatics force field ( 21 ) , a poisson boltzmann - optimized force field by tan et al . user - defined parameters can be uploaded to the pdb2pqr server in a simple flat - file format described in the pdb2pqr user guide . additionally , pdb2pqr output can be customized to include a variety of atom naming schemes , including amber99 ( 19 ) , charmm22 ( 20 ) , parse ( 21 ) and an internal naming scheme based on the iupac naming recommendations ( 23 ) . this flexibility in nomenclature was included to facilitate import of pdb2pqr output into other modeling packages . map which is output at the end of every pdb2pqr calculation and presents a table of atoms name / number , residue name , chain name , amber atom type and charmm atom type to aid in the interpretation of parameter assignment and the development of user - defined charges and radii . the calculation of ligand charges necessitates detailed information on molecular structure and protonation states due to the large variation in the covalent structures of small - molecule protein ligands . the current version of pdb2pqr therefore requires the ligand structure , protonation state and formal charge to be specified by the user in the popular mol2 ( 24 ) format . ligand structures in mol2 format are readily available from popular molecular modeling software and free web services such as prodrg ( 25 ) . future versions of pdb2pqr will include a pdb2mol2 parser and automatic assignment of default ligand protonation states from a small - molecule pka database . the calculation of ligand charges in pdb2pqr is based on the partial equalization of orbital electronegativities ( peoe ) procedure developed by gasteiger and marsili ( 26 ) . in the peoe procedure , orbital electronegativities are linked to partial atomic charges q by a polynomial expansion ( = a + bq + cq + dq ) . the coefficients a , b , c and d were optimized by gasteiger and marsili using gas phase data on ionization potentials and electron affinities . we utilize a peoe algorithm , which has been optimized by czodrowski et al . to obtain better agreement between theoretical and experimental solvation energies for a set of small molecules including the polar amino acids ( 27 ) . the resulting peoe_pb charges have been tested for small - molecule complexes with trypsin , thrombin ( 28 ) and hiv protease ( 29 ) , and have been found to give results that are in agreement with experimental values . such extensions might include alternative naming schemes , identification and parameterization of other molecule types , additional hydrogen bond processing , etc . however , it is straightforward for users to download the pdb2pqr software and setup their own web servers with additional functionality based on custom extensions . we have described a number of new features for the free pdb2pqr web server , a service which helps users prepare molecular structures for further computational work by modeling missing atoms , assigning charges and titration states , and providing a mechanism for assignment of ligand parameters . readers interested in these tasks might also be interested in other servers , which provide complementary services for biomolecular structure processing ( 3032 ) . planned future developments for pdb2pqr include the construction of a pdb2mol2 parser to allow for the automatic parameterization of non - protein atoms , the correct treatment of protein post - translational modification , and the integration of a poisson we anticipate that the pdb2pqr service will continue to be a helpful addition to the portfolio of tools available to the structural and computational biology communities .

family planning ( fp ) is a process that usually involves a discussion between a woman , a man , and a trained fp service provider focusing on family health and the desires of the couple to either limit or space their family . these are long - acting and permanent methods ( intrauterine devices , implants , and sterilization ) and short - term methods ( pills , condoms , spermicides , injectables , other modern methods , and all traditional methods ) . long - acting and permanent methods are usually used to limit childbearing , whereas short - term methods are better suited for women who want to delay but not forfeit having a child a great majority of the health facilities in ethiopia offer oral pills ( 98.8% ) and injectables ( 98.0% ) followed by male condom ( 95.2% ) , implants ( 75.0% ) , iuds ( 53.6% ) , female sterilization ( 22.6% ) , male sterilization ( 16.7% ) , and female condom ( 4.0% ) . thirty - seven percent of women want no more children but only 3.4% of married women reported using implants , 0.3% iud , and less than 1% as sterilized . the overall prevalence of long acting and permanent contraceptive methods ( lapms ) was 12.3% . majority of the participants ( 93.3% ) stated that using another method of contraception is their main reason for not using lapms . a significant amount of the participants had low knowledge on permanent contraceptive methods , particularly vasectomy . more than half ( 53.6% ) of married women had negative attitude towards practicing of lapms . they said it might cause excessive bleeding , infertility , or cancer [ 6 , 7 ] . there was a widespread belief that the intrauterine device ( iud ) makes the user more prone to sexually transmitted infections ( stis ) and infections of the pelvis . some women perceive that implants could get lost in the body via the blood stream . similarly , they perceive that iud could shift during sexual intercourse with serious implications for birth outcomes . information on women 's and men 's knowledge on contraceptive methods provides a measure of awareness of contraception in the population and indicates the success of information , education , and communication programmes . in addition , knowledge of at least one modern contraceptive method and a positive attitude towards that contraceptive method are prerequisites for the use of that contraceptive . the objective of this study was to determine knowledge and perception regarding lapms among married women and men . the findings of this assessment will help in designing relevant and culturally appropriate interventions that will aid the efforts at promoting and disseminating information on lapms to the eligible individuals and couples . four focus - group discussion ( fgd ) sessions with purposively selected three groups of married women and one group of married men were conducted . separate male and female fgds were seen as the preferred protocol in order to discuss fp and reproductive health , which are considered private and sensitive topics . a purposive sampling approach was employed to recruit 32 participants of the target group consisted of seven up to twelve discussants . both men and women had been identified and the participants were recruited through community leader contacts . the most commonly used form of nonprobabilistic sampling is purposive sampling and their size relies on the concept of saturation , or the point at which no new information or themes are observed in the data or cutoff between adding emerging findings and not adding or when the researcher is no longer hearing or seeing new information . the inclusion criteria were married men and women , with ages between 15 and 49 years for women and 18 and 65 years for men . fgd guide was used for eliciting information including the following topics such as social , demographic issues , knowledge and practice of modern contraceptives , method preference , advantages and disadvantages of lapms , barriers and perceptions about fp , community attitude and perception concerning the use of lapms , and suggestions . a total of six in - depth interviews were conducted with purposively selected family planning ( fp ) service providers to see the perceptions and choice of clients towards lapms . the inclusion criterion was health professionals in the health facilities who offer any of the lapms and were working during the data collection period . interview guide was used to extract information about training status of the provider ; capacity of counseling and providing long acting and permanent contraceptive methods ; women 's knowledge , perception , and preference of modern contraceptives in their clinic ; what could improve the provision of family planning services in particular lapms ; and suggestions . audio data were transcribed verbatim into microsoft word files and translated from the local language to english . the principal investigator and note taker reviewed key terms in local language and their respective translations to ensure a degree of standardization . a small , selected sample of interviews was translated from tigrigna into english by an independent translator to assess the accuracy of the translation . final transcripts were compared against note takers ' notes to ensure quality . before the analysis , the text was read through several times to obtain a sense of the whole and familiarize with the data . the data was first saved in text format and imported in to open code software version 3.6.2.0 to facilitate coding and categorizing . the various codes were compared based on differences and similarities and sorted into categories . finally based on content analysis , the underlying meaning that is the latent content of the text the results contain direct quotes from participants and narrations are reported as spoken by participants without editing the grammar to avoid losing of the meaning . quotes that best described the various categories and expressed what was said frequently in several groups were chosen . ethical clearance was obtained from research and ethics committee of school of public health , college of health science of addis ababa university . written permission letter was also produced from tigray regional health bureau and then from adigrat woreda health office . twenty - three of the participants were modern contraceptive users , which was dominated by depo - provera ( table 1 ) . six in - depth interviews with fp service providers in public and private health institutions were conducted . majority of the participants in the fgd were able to identify short - term ( depo - provera , pills , and condom ) and long acting contraceptive methods ( implants and iud ) . but most of the participants were not able to identify permanent contraceptive methods ( male and female sterilization ) as contraception . both women and men in the fgd lack specific information on how lapms work especially permanent methods of contraception . i did not hear about sterilization of female as well as male before . ( 30 years old woman , illiterate , para five , depo - provera user ) i heard the presence of medication which is inserted in to hand . ( 30 years old man , grade 7 completed ) i am government employee . i never learnt before as today , because nobody teaches us about such things ( 37 years old man , diploma ) i did not hear about sterilization of female as well as male before . ( 30 years old woman , illiterate , para five , depo - provera user ) i never learnt before as today , because nobody teaches us about such things ( 37 years old man , diploma ) the in - depth interviewees indicated that the knowledge of the women on lapms is superficial , limited to identifying the methods name . they said that the quality of counseling service they provide is not inclusive of all contraceptive choices . we ( health providers ) do not explain well to them on each contraceptive method . we ( health providers ) do not explain well to them on each contraceptive method . they had concern about negative effect on the return of fertility after taking implants or iud . some women had expressed their concern on iud on the need of vaginal examination , discomfort during sex , side effects , and lack of protection against sexual transmitted infections . implant has problem in your daily activity . ( 24 years old woman , 9th grade completed , depo - prevera user ) implant has difficulty in doing hard works which results to numbness of your hand . implant has a problem with work especially it is not good for women living in the village working a lot . ( 28 years old woman , 4th grade completed , implanon user ) implant has problem in your daily activity . ( 24 years old woman , 9th grade completed , depo - prevera user ) implant has difficulty in doing hard works which results to numbness of your hand . implant has a problem with work especially it is not good for women living in the village working a lot . ( 28 years old woman , 4th grade completed , implanon user ) they told stories about difficulty of removal and resistance to remove implants by the fp providers . they ( fp providers ) did not remove timely at the time you want to remove . i know one lady ; she is my neighbor , she had visited them repeatedly for removal . she had suffered because they did not obey to remove while she wants to remove it . ( 36 years old woman , illiterate ) they ( fp providers ) did not remove timely at the time you want to remove . i know one lady ; she is my neighbor , she had visited them repeatedly for removal . she had suffered because they did not obey to remove while she wants to remove it . ( 36 years old woman , illiterate ) the fp providers stated that there are fears and rumors raised by the women about lapms . most of the customers had fear of procedures during insertion and removal and fertility return after the use of implants and iud . long acting is perceived by the mothers as it needs surgical procedure ( operation ) . implant can cause numbness of the hand , it can pierce you , and you ca n't work while the implant is in your hand . they perceive iud as it can go to your head , and not good during sex . i face only one male who come for sterilization and he returned back because we have no service here . they know only pill and injection but even though implant last long ; they consider as new thing . ( idi 01 ) long acting is perceived by the mothers as it needs surgical procedure ( operation ) . implant can cause numbness of the hand , it can pierce you , and you ca n't work while the implant is in your hand . they perceive iud as it can go to your head , and not good during sex . i face only one male who come for sterilization and he returned back because we have no service here . they know only pill and injection but even though implant last long ; they consider as new thing . ( idi 01 ) the fgd and in - depth interview result revealed that depo - provera and pills were considered as normal contraceptive methods . specifically , injection ( depo - provera ) was perceived as easy to use and stop , easy accessible , and free from any procedures . they did not need procedure and support from others to stop or remove like implant and iud . ( idi 02 ) me myself i have used injection ( depo - provera ) for seven years , i did not face any problem . they did not need procedure and support from others to stop or remove like implant and iud . ( idi 02 ) me myself i have used injection ( depo - provera ) for seven years , i did not face any problem . ( 38 years old woman , illiterate , depo - provera user ) most of the participants in the in - depth interview stated that mothers prefer short - term methods ( depo - provera and pills ) . the reason they had stated is that the quality of counseling service provided by the fp providers is not inclusive of all contraceptive choices . the fear of procedures for lapms is another reason pushing mothers to prefer short - term methods of contraceptives . they have fear of resistance by the fp providers to remove the long acting methods . first the mother should be counseled well , she should also have freedom of decision on removal . but what they usually complain is bleeding , body weakness , hair loss , behavior change because of the implant . those who took injection when we counsel about long acting contraceptives , they said i will think for that latter but for now i should take depo - provera . ( idi 03 ) injection is considered as normal because it is well known whereas long acting is new ; but it will be popular as injection in the future . therefore , if it is introduced well ; it will be used by everybody . they have fear of resistance by the fp providers to remove the long acting methods . first the mother should be counseled well , she should also have freedom of decision on removal . but what they usually complain is bleeding , body weakness , hair loss , behavior change because of the implant . those who took injection when we counsel about long acting contraceptives , they said i will think for that latter but for now i should take depo - provera . injection is considered as normal because it is well known whereas long acting is new ; but it will be popular as injection in the future . therefore , if it is introduced well ; it will be used by everybody . family planning providers in the in - depth interview indicated that the counseling service they were providing was not inclusive of all modern contraceptives . women , visiting the clinic , had a preference for a type of contraception and they provide what women asked for . they had also indicated the need of training of a team of health providers on lapms . in our clinic we have provided a lot of long acting contraceptives , almost one per day for long acting contraceptives . i want to say that the health professionals should educate the community with the focus on long acting contraceptives . if you educate appropriately they can easily change and use long acting contraceptives . because of knowledge women prefer short term but we should not give her simply what she say ( idi 041 ) all professionals should get training on long acting contraceptives . otherwise there is pressure on one provider to do all the counseling , insertion and removal . this may not be effective . if every health professional have got training on these methods ; the workload will be shared and clients will be served well . ( idi 042 ) there is no good knowhow , we are too much busy . we have problem in counseling because of the work load otherwise if we properly counsel the women ; they can choose long acting contraceptives . i recommend that the community leaders and religion leaders can support in teaching the whole community . ( idi 05 ) in our clinic we have provided a lot of long acting contraceptives , almost one per day for long acting contraceptives . i want to say that the health professionals should educate the community with the focus on long acting contraceptives . if you educate appropriately they can easily change and use long acting contraceptives . because of knowledge women prefer short term but we should not give her simply what she say they educate women and they bring to our facility for the service . ( idi 041 ) all professionals should get training on long acting contraceptives . otherwise there is pressure on one provider to do all the counseling , insertion and removal . this may not be effective . if every health professional have got training on these methods ; the workload will be shared and clients will be served well . ( idi 042 ) there is no good knowhow , we are too much busy . we have problem in counseling because of the work load otherwise if we properly counsel the women ; they can choose long acting contraceptives . i recommend that the community leaders and religion leaders can support in teaching the whole community . they lack knowledge on how these methods work and how they can use them especially concerning permanent methods of contraception . they had expressed their concern on the return of fertility after using implants or iud . they had fear of resistance of the providers on removal of implants up on their demand to remove . the fp providers indicated the presence of fears and rumors , raised by their clients about each method , and mistrust towards the providers on removal especially implants . the fp counseling service is not comprehensive of all modern contraceptive methods , especially lapms . short - term contraceptives ( depo - provera and pills ) were the most commonly known and preferred methods . this avoids the fear of resistance to remove implants and iud by the fp providers . but the inclusion of lapms especially permanent methods ( tubal ligation , and vasectomy ) in the counseling of methods was still not well done . in interpreting these results , there are certain limitations with the study design that might impact upon the conclusions drawn . first and foremost , the purposive sampling may be biased by including individuals who are more comfortable talking about family planning matters . besides other acknowledgeable limitations associated with the use of qualitative methodology , the result may not be automatically transferred to other countries with different cultural contexts . the strength of this study lies in its richness of data , including women , men , and fp providers , which ensured enough data triangulation and saturation . knowledge on different contraceptive methods is the key to choose the contraceptive method and to practice it [ 11 , 12 ] . almost universal awareness of at least one modern contraceptive method was observed in the studies conducted in southern ethiopia [ 13 , 14 ] . but this finding may have been the result of knowledge of a method variable that was evaluated as have you heard about it , which may not actually reflect to an adequate knowledge to the method in question . permanent contraceptive methods were the least known method in the ethiopian demographic health survey 2011 and study conducted in mekelle [ 4 , 5 ] . some women perceived that contraceptives interfered with fertility , and they were scared to use something that could harm their ability to reproduce [ 7 , 1518 ] . the experiences of other people on specific contraceptive method strongly affect the choice and perception of other women . more than one quarter ( 29.7% ) of married women perceive that iud insertion procedure can result in shame . some women perceive iud as it can limits day to day activities , and female sterilization is dangerous . negative provider attitudes , misinformation on the part of providers and high service charges were identified as barriers to fp use in pakistan . every woman who seeks fp information or services should be educated and counseled on all choices of modern contraceptive methods ; and given an opportunity to ask questions after the provider has described the methods available . educational and enlightenment programs should focus on providing specific knowledge , with special attention to correcting common misconceptions about the methods . health care providers should be encouraged to inform all potential users about the methods , and to prescribe it to clients who require it . this study is carried out to explore the knowledge and perceptions of married women and men on lapms . knowledge of specific methods is very low particularly of long - term and permanent methods of contraception . therefore , there is need to broaden the knowledge of such methods of family planning . improving the counseling service program can help women to increase the knowledge of each contraceptive choice and choose appropriate contraceptive methods and use them consistently . women need more information from their health care providers in order to make informed choices about their fp preferences . therefore , the fp providers should counsel on all of the fp methods , address misconceptions and fears that exist about lapms , and highlight the benefits of lapms during fp counseling . the fp counseling should also include the benefits , mechanism of action of modern contraceptive methods , and side effects and how to manage them . teams of health care workers should be trained to offer the service , not just individuals within health centers and clinics . further detailed investigation of the quality of counseling service in the town should be conducted .

olive ( olea europaea ) is in the family oleaceae that is used throughout the world especially in the mediterranean region . olive oil is rich in monounsaturated fatty acids ( mufas ) and has excellent health benefits . olive oil is composed mainly of mixed triglyceride esters of oleic acid , palmitic acid , and other fatty acids , along with the traces of squalene ( up to 0.7% ) and sterols ( about 0.2% ; phytosterol & tocosterols ) . it also contains group of antioxidants that are esters of tyrosol and hydroxytyrosols , including oleocanthal , oleuropein , vitamin e , and carotenoids . oleuropein is a chemical that prevents the oxidation of ldl ( low density lipoproteins ) particles . evidence from epidemiological studies suggests that a higher proportion of monounsaturated fats in the diet are linked with a reduction in the risk of coronary heart disease . it has been reported that olive oil consumption has favorable effects on cholesterol regulation and ldl cholesterol oxidation and it also exerts antiplatelet , anti - inflammatory , antithrombotic , and antihypertensive as well as vasodilatory effects on both animals and humans . phenolic compounds , oleic acid , and tocopherols are also thought to be related to some of these beneficial effects . olive oil could possibly be a chemopreventive agent for peptic ulcer or gastric cancer and was found to reduce oxidative damage to dna and rna , which may be a factor in preventing cancer . olive oil is rich in omega 3 and omega 6 fatty acids which help to improve memory and support the cns by aiding nerves to do proper function and increases serotonin levels . evidence has shown that high mufa intake appeared to be protective against age - related cognitive decline and helps to aid the memory . olive oil also helps to build a more healthy balance between omega-6 fats and omega-3 fats . it has been reported that omega-3 fatty acid , docosahexaenoic acid ( dha ) , plays an adaptive role during stress and shows a significant reduction in perceived stress by increasing secretion of adrenal corticosterone in rats loaded with single repetitive stress . oleuropein derivatives found in olive oil especially hydroxytyrosol have been shown to have protective effects not only against markers associated with antherogenic process [ 7 , 15 , 16 ] but has an antioxidant capacity higher than that of other known antioxidants such as vitamins e and c . in addition to this , an anxiety lowering effect with reduced step - through latency in light dark box test was also found in old animals that the effect was due to the decreased glutathione reductase activity and expression in the brain . studies have also reported that treatment with olive extract resulted in a significant decline in the immobility time as well as hyperalgesia . it is evident that oleamide induced increase in slow - wave sleep , a decrease in wakefulness and sleep latency , was prevented by 5-ht reuptake inhibitors such as fluoxetine and by agonists at 5-ht1a receptors such as buspirone or 8-oh - dpat . oleamide causes a potentiation of 5-ht elicited inositol phosphate formation mediated by the 5-ht2a receptors but inhibited the effects of 5-ht on camp production mediated by 5-ht7 receptor . hence , extravirgin olive oil is becoming more important in daily diets nowadays , particularly in mediterranean diet , due to its beneficial health effects on human health . therefore , the present study was designed to investigate the neuroprotective effects of extra - virgin olive oil in rats and to elucidate the neurochemical changes associated with it . male albino wistar rats weighing from 140280 g were purchased from animal house of hej research institute of chemistry , university of karachi , pakistan . the animals were caged separately in plastic cages at room temperature of 22 2c with free access to tap water and cubes of standard rodent diet during the whole session of experiments . before starting each experimental session , rats were accustomed to various handling procedures in order to avoid any stress effect and to nullify the psychological affliction of the environment . all the experiments were performed ethically in accordance with the guidelines of the local animal care ethical committee . control rats received water while to the test group olive oil was given at the dose of 0.25 ml / kg body weight daily for 27 days . rats were then decapitated , and their brain was removed within 30 seconds from skull . frozen brain samples were homogenized in extraction medium using an electrical homogenizer , and neurochemical analysis was performed to estimate concentrations of 5-ht ( 5-hydroxytryptamine ) and its metabolite 5-hiaa ( 5-hydroxyindoleacetic acid ) and da ( dopamine ) and its metabolite hva ( homovalinic acid ) in the whole brain of rats by hplc - ec method as reported by haider et al . 2004 . a 5 shim - pack ods separation column of 4.0 mm internal diameter and 150 mm length was used as the stationary phase . separation was achieved by a mobile phase containing methanol ( 14% ) , octyl sodium sulfate ( 0.023% ) , and edta ( 0.0035% ) in 0.1 m phosphate buffer at ph 2.9 at an operating pressure of 20003000 psi on schimadzu lec 6a detector at an operating potential of 0.8 volts for biogenic amines . elevated plus maze ( epm ) is an apparatus used to evaluate the rodent 's natural behavior which is fear of novel and open areas . the anxiolytic activity of drug in elevated plus maze model was measured according to method as previously reported . plus the length of each arm was 50 cm and width is 10 cm . the maze was elevated from ground at a height of 60 cm . to determine activity , a rat was placed in the center of the plus maze and the time spent in the open arm was monitored for 5 minutes . assessment of depressive symptoms was monitored by forced swim test ( fst ) following 4 weeks of oral administration of drug . to monitor the antidepressant activity , rats are placed individually in a tank ( 53 , 19 , 28 cm ) . the animal is subjected in the container for 5 minutes , and behavioral scoring was performed by noting immobility time . after each test , rats were dried with towel and placed in home cage . values are represented as mean sd , and values of p < 0.05 are considered as significant . figure 1 shows the effect of repeated administration of olive oil on depression like symptoms in animals . student 's t - test analysis showed that there is a significant ( p < 0.05 ) increase in struggling time following repeated administration of extravirgin olive oil indicating that olive oil has significant antidepressant effects . analysis by student 's t - test revealed that there was a significant ( p < 0.05 ) increase in time spent in open arm following repeated administration of extra - virgin olive oil . table 1 shows the effect of olive oil on levels of brain 5-ht and its metabolite 5-hiaa . student 's t - test analysis showed a significant ( p < 0.05 ) decrease in brain 5-ht and 5-hiaa levels following repeated administration of extra - virgin olive oil which indicates a decrease in 5-ht metabolism and synthesis . table 2 shows the effect of repeated administration of olive oil on levels of brain da and its metabolite hva . analysis by student 's t - test revealed that there was a significant ( p < 0.05 ) decrease in brain da levels while the hva levels were significantly ( p < 0.01 ) increased following repeated administration of extra - virgin olive oil which indicates that olive oil administration led to an enhancement in hva levels and a decrease in brain da by increasing da metabolism . olive oil is used throughout the world mostly in the mediterranean because of its beneficial health effects . in the last few years recent data has suggested that component of olive oil may have more health benefits than previously thought . the present study reveals that repeated administration of extra - virgin olive oil produces anxiolytic and antidepressant effects via neurochemical alterations in brain 5-ht , da , and their metabolites . reports have shown that low levels of omega-3 and omega-6 fatty acids are involved in producing depression as omega-3 fatty acids are important component of nerve cell membrane and help nerve cells to communicate with each other , which is an essential step in maintaining good mental health . omega-3 fatty acid of dha group has been shown to decrease or prevent aggression during examination . high levels of omega-3 fatty acid in serum have been shown to lower the levels of pro - inflammatory cytokines thus protecting from stress . in the present study , it is has been observed that repeated administration of extra - virgin olive oil produce significant antidepressant effects as the struggling time of rats taking olive oil was significantly increased as compared to control rats . this indicates that olive oil has great potential to reduce depression which may be due to presence of linoleic acid ; a polyunsaturated omega-3 fatty acid that makes up 1.5% of extra - virgin olive oil . evidence from animal and human studies showed that da and na play important role in pathophysiology as well as in therapeutic effect of antidepressants . evidence from clinical studies supports the finding that depressed patients have reduced csf ( cerebrospinal fluid ) levels of hva . decreased da and increased levels of hva are an indication of increased release of da and increased turnover which may play important role in antidepressant effect of da . decreased da and increased hva levels together with the increased struggling time following repeated administration of extra - virgin olive oil observed in the present study support the findings that olive oil produces antidepressive effect by increasing the release and turnover of da . the present findings also revealed that the anxiolytic property of extra - virgin olive oil as time spent in open arms in epm was increased significantly following repeated administration of extra - virgin olive oil . the anxiolytic effect of extra - virgin olive oil could be due to presence of phenolic antioxidants , olieic acid , tocopherols , and oleuropin in olive oil . anxiolytic effects of olive oil observed in the present study may be due to alterations in brain 5-ht metabolism . increased 5-ht produces anxiogenic effect while decreased level of 5-ht produces anxiolytic effect . in the present study , it has been found that extra - virgin olive oil decreases brain 5-ht as well as 5-hiaa levels in rat brain . administration of extra - virgin olive oils ignificantly decreases 5-ht synthesis and metabolism that may play important role in reducing anxiety in animals . decreased 5-ht and 5-hiaa levels together with the increased time spent in open arm in epm observed in present study olive oil has important effect on body and has protective effect against several pathological conditions . results of present study show that olive oil produces anxiolytic effect via decreasing brain 5-ht synthesis and metabolism . however , antidepressive effect of olive oil may attribute to the increased da release and its metabolism . results of the present study supported the use of olive oil as food supplementation for mood elevation .

acne inversa ( ai , also referred to as hidradenitis suppurativa ) is a chronic inflammatory skin disease with common onset in the second or third decade of life [ 1 , 2 ] . estimates of the prevalence of ai have varied , ranging from less than 1 percent to 4 percent [ 3 , 4 ] . the axilla and the inguinal , gluteal , and perianal area are the most common affected sites . the inner thighs , perineal areas , sub- and inframammary skin , and gluteal cleft are additional sites for involvement [ 1 , 2 ] . the clinical manifestation progresses from nodules and inflamed lesions with deep abscesses to chronic , draining sinus tracts and bands of severe scar formation . the associated pain , large amount of purulent exudate , malodor , and disfigurement contribute to a profound psychosocial impact of the disease on affected patients [ 3 , 5 , 6 ] . additionally , ai is frequently associated with metabolic alterations that might increase the risk of cardiovascular disorders and reduce the life expectancy [ 7 , 8 ] . an early and accurate diagnosis would allow the initiation of a treatment plan aimed at minimizing the risk of progression to disabling , end - stage disease . however , as of today , on average 12 years pass between first symptoms and accurate diagnosis . the current model implies that the follicular occlusion of the pilosebaceous unit by infundibular hyperkeratosis leads to dilatation and rupture of the unit as initial steps . at very early stages of the disease chronic ai lesions feature immune cell infiltration and strong expression of numerous proinflammatory cytokines like tnf- and il-17a . the inflammation seems to increase the destruction of skin architecture and the development of deep fistulating sinuses . currently , the disease severity of ai can be assessed by using two clinical scores . the hurley classification is a simple system that divides ai into three severity grades for each area involved . however , it is static and not suitable for the assessment of inflammatory aspects of the disease . secondly , the sartorius score with its modified versions [ 15 , 16 ] is a dynamic score that takes into account numerous parameters including the number of affected regions , the type of cutaneous alteration , lesion diameters , and the presence of healthy skin between lesions . the sartorius score yields a count between zero ( inactive disease ) and about 250 . it requires some experience and its alterations may result from changes in various aspects of the disease . furthermore , to a certain extent both scores lack objectivity , with the final evaluation being dependent on the examiner 's interpretation of the physical findings . in our study we aimed to investigate the possible suitability of mmp8 as blood biomarker that could be exactly and easily quantified and help to assess disease activity in ai . furthermore , we wanted to explore the cellular source of mmp8 as well as its role in ai pathogenesis . skin biopsies for gene expression analyses by means of polymerase chain reaction on reversely transcribed rna ( rt - qpcr ) were obtained from 8 control participants , 10 patients with ai , and 10 patients with psoriasis . the skin biopsies of ai patients were taken from surgically excised skin areas and such biopsies were also used for immunohistochemistry ( ihc ) . blood plasma and serum were obtained from 20 control participants [ 27 to 57 years old ( mean sd : 39.0 9.3 years ) , 25% male ] and from 21 patients with ai [ 22 to 56 years old ( mean sd : 41.7 11.6 years ) , 10% male , sartorius score : 52.9 40.9 ( mean sd ) ] . there were no significant differences in age or sex distribution between control participants and ai patients . all skin and blood samples were approved by the clinical institutional review board of the charit university hospital , berlin , and written consent was obtained from all participants . human blood was diluted 1 to 5 with rpmi medium and stimulated or not ( control ) with 10 ng / ml ifn- , 5 ng / ml tnf- , 5 ng / ml il-1 , 10 ng / ml il-6 , 10 ng / ml il-17a , or 20 ng / ml il-22 for 4 h to activate neutrophilic granulocytes . primary dermal fibroblasts ( lonza / life technologies / promocell ) and primary human keratinocytes ( lonza ) were cultured in fgm-2 medium and kgm - gold medium , respectively ( both from lonza ) , according to the suppliers instructions and as previously described [ 17 , 18 ] . fibroblasts were stimulated or not ( control ) with 10 ng / ml ifn- , 5 ng / ml tnf- , 10 ng / ml il-17a , 50 ng / ml il-19 , or 20 ng / ml il-24 for 24 h. keratinocytes were stimulated or not ( control ) with 10 ng / ml tnf- , 10 ng / ml il-17a , 10 ng / ml il-22 , or indicated combinations thereof for 6 h. all cytokines mentioned above were purchased from r&d systems . tissue homogenization , isolation of total cellular rna , mrna reverse transcription , and qpcr analysis were done as previously described [ 19 , 20 ] . expression values were normalized to abundance of mrna transcripts of the house - keeping gene human hypoxanthine - guanine phosphoribosyltransferase 1 ( hprt ) . detection of human mmp8 ( lithium heparin plasma ) , timp-4 ( lithium heparin plasma ) , tnf- ( serum ) , and resistin ( edta plasma ) was performed using quantikine systems from r&d systems . quantification of plasma lipids was carried out using cobas 8000 modular analyzer series , roche diagnostics gmbh . skin biopsies were fixed in 10% neutral buffered formalin , embedded in paraffin , routinely processed , sectioned at 5 m , and stained with antibodies ( ab ) recognizing myeloperoxidase ( dako ) . detection of primary ab binding was performed using biotinylated polyclonal rabbit anti - rat igg , streptavidin horseradish peroxidase and aec+ substrate ( all from dako ) , or the lsab2 system - hrp kit ( dako ) ; counterstaining was done with hematoxylin . data are presented as the mean sem . for further analyses , spss 19.0 software ( ibm ) results from patients / control participants were analyzed using the mann - whitney u test ( two - tailed ) . results on primary cell cultures were tested using the wilcoxon matched - pairs signed - rank test ( two - tailed ) . the mechanisms associated with the development and progression of ai lesions ( figure 1(a ) ) are still enigmatic . to identify potential key players in these processes , we first individually quantified the expression of a broad range of immune parameters in affected skin of ai patients and skin from control participants . from 25 investigated mediators [ il-4 , il-6 , il-13 , il-20 , il-25 , il-33 , il-34 , il-12 p35 , il-23 p19 , p40 , ifn- , gm - csf , ccl2 , ccl8 , ccl17 , ccl20 , ccl22 , ccl26 , cxcl2 , cxcl9 , cxcl10 , cxcl11 , -defensin ( bd)-1 , bd3 , metalloproteinase ( mmp ) 8 , and cd54 ] , mmp8 was the parameter with the highest upregulation in ai lesion compared to skin from control participants . mmp8 ( also referred to as collagenase 2 ) is an enzyme specialized in the degradation of extracellular matrix components . the elevated expression of mmp8 in lesional ai skin may therefore be involved in the development of draining sinus tracts and the destructive character of this disease . interestingly , increased mmp8 expression was specific for ai lesions and not observed in affected skin of patients with psoriasis , a chronic inflammatory skin disease without sinus tract development ( figure 1(b ) ) . mmp8 is known to be stored in secondary granules within neutrophilic granulocytes and secreted in its active form . because marked presence of neutrophilic granulocytes was found in ai lesions ( figure 2(a ) ) , we firstly investigated different ai - relevant cytokines for their ability to induce mmp8 secretion from neutrophilic granulocytes . as demonstrated in figure 2(b ) , it was predominantly tnf- and , to a lesser extent , il-1 that raised the release of mmp8 in whole blood assay . this tnf- effect could not be amplified by il-17a . also , we investigated whether tnf- can as well induce mmp8 expression in skin - resident tissue cells . interestingly , dermal fibroblasts but not epidermal keratinocytes showed mmp8 expression after tnf- stimulation ( figures 3(a ) and 3(b ) ) . activation of keratinocytes with a mix of tnf- , il-17 , and il-22 , a very potent activator for these cells , did not result in significant mmp8 mrna expression either ( figure 3(b ) ) . then , we asked whether the high mmp8 levels in lesional ai skin were accompanied by elevated systemic levels . the analyses of blood samples from 21 ai patients and 20 control participants disclosed a significant increase of mmp8 in the blood plasma of ai patients ( figure 4 ) . interestingly , there were no differences in the blood levels of tissue inhibitor of matrix metalloproteinases ( timp ) 4 between ai patients and control participants ( figure 4 ) . the analysis of potential relationships of mmp8 blood levels with clinical symptoms revealed that mmp8 blood levels positively and significantly correlated with ai severity assessed by sartorius score ( table 1 ) . further analyses revealed an association between mmp8 blood levels and the number of regions with inflammatory nodules and fistulas , but not with scars ( table 1 ) . there was no correlation between mmp8 blood levels and duration of illness , age at ai onset , or patient age ( table 1 and data not shown ) , suggesting that mmp8 levels are associated with the inflammatory activity in this disease . to get hints for further roles of mmp8 in ai , we analyzed levels of several mediators in blood of ai patients and correlated them with mmp8 blood levels . as demonstrated in figure 5(a ) , a clear positive association between mmp8 and tnf- blood levels was observed , matching very well our results showing tnf- as key mmp8 inducer ( figures 2(b ) and 3(a ) ) . interestingly , we also found a specific negative correlation between mmp8 and high - density lipoprotein ( hdl ) cholesterol blood levels ( figure 5(b ) ) . furthermore , there was a positive association between levels of mmp8 and resistin ( figure 5(c ) ) , a cysteine - rich adipose tissue - derived peptide hormone that accelerates the accumulation of ldl in artery walls . overall , the last data suggest a pathogenetic role of mmp8 in the metabolic alterations of ai patients . it destructs axillary , inguinal , gluteal , and perianal regions of very young people , causing profound pain , large amounts of purulent and malodorous exudate , and metabolic alterations , and results in reduced quality of life , social isolation , and diminished life expectancy . evidence for pathogenetically relevant molecules that at the same time are useful as blood biomarkers for more exact quantification of ai disease severity may facilitate the development of novel therapies for these patients . we show a strong upregulation of mmp8 in ai lesion compared to skin from healthy controls and psoriasis patients . mmp8 is mainly produced by neutrophilic granulocytes and this very well matches the observed strong dermal infiltration of al lesions by neutrophils and the large purulent exudates in these patients . in contrast , neutrophilic infiltration is limited to epidermal munro 's microabscesses in psoriasis , and purulence exudates are absent in this disease . regarding mmp8 production , we additionally demonstrated that tnf- is a potent inductor of mmp8 in neutrophilic granulocytes and , moreover , exerts this effect also on fibroblasts . it is possible that bacterial products induce tnf- production in lesional monocytes , macrophages , and neutrophilic granulocytes and contribute to elevated mmp8 expression . the major substrate of mmp8 is the collagen type 1 that forms large collagen fibers in most connective tissues , including the dermis , giving these tissues rigidity and elasticity [ 21 , 23 ] . we assume that degradation of this extracellular matrix component by mmp8 in ai lesions might lead to the development of skin cavities ( abscesses and draining sinus tracts ) , as very recently demonstrated for cavities developing in lung tissue during human pulmonary tuberculosis . due to the lack of cutaneous mmp8 expression in psoriatic lesions , abscesses and draining sinus tracts are absent in psoriasis . in the last part of our study we demonstrated that the high lesional mmp8 levels in ai were accompanied by elevated mmp8 blood levels . importantly , these levels positively correlated with disease activity , in particular with their inflammatory components . the currently used methods for evaluating the disease activity are often time - consuming ( especially with sartorius ' grading system ) and undoubtedly subjective ; deviations commonly occur when assessment is conducted by different physicians and centers . what is currently needed are laboratory parameters useful for accurate ai staging , for decision of the therapeutic regimen , and for assessment of treatment efficacy . interestingly , our study also revealed an association between mmp8 and metabolic alterations in ai patients . in fact , mmp8 blood levels negatively correlated with hdl - cholesterol , an antiatherogenic lipid . accordingly , salminen at al . described that mmp8 affects the structure and antiatherogenic function of apolipoprotein ( apo)a - i , the main protein component of hdl particles . proteolytic modification of apoa - i by mmp8 has been found to impair the first steps of reverse cholesterol transport , leading to increased accumulation of cholesterol in blood vessel walls . it stimulates proinflammatory cytokine expression in human monocytic cells and increases adhesion molecule expression and chemokine production by endothelial cells [ 27 , 28 ] , leading to immune cell extravasation and development of atherosclerotic plaques . in summary , our results demonstrate the strong presence of mmp8 in skin lesions and blood of ai patients and suggest the pathogenetic involvement of this molecule in skin and systemic alterations observed in ai patients .

diabetes mellitus , a metabolic disorder , results from an inadequate mass of insulin - producing pancreatic beta cells [ 1 - 3 ] . various formulations of short- and long - lasting insulin have been used to control blood glucose levels . however , the tight regulation of insulin in response to physiological change is not possible . the development of a therapeutic method to regulate precisely the blood glucose levels will enable better management of diabetes . islet transplantation into diabetic patients is a promising method for restoring the functional beta cell mass . however , the limited supply of islets can not meet the patient demand , and post - transplant immunosuppression can produce serious side - effects . to overcome these limitations , various methods providing an alternative source of insulin - producing cells are being investigated . some of these methods include engineering non - beta cells to produce insulin , differentiation of insulin- producing cells from embryonic and adult stem / progenitor cells , and transdifferentiation of extra - pancreatic and pancreatic cells . in addition , in vivo regeneration of islet cells is being investigated . in this review , we will provide recent advances , as well as research progress on beta cell replacement and regeneration therapy for treatment of diabetes using various strategies ( fig . islet or pancreas transplantation is a method to normalize metabolic control in a way that can not be achieved with exogenous insulin . it was shown that whole pancreas transplantation ameliorated the complications associated with chronic hyperglycemia and eliminated the need for daily insulin injections . however , pancreas allografting caused immediate surgical risks and long - term complications . the first trial of islet transplantation was performed in rodent models and later in type 1 diabetic patients . further advances in the islet isolation procedure and more effective immunosuppression strategies with a steroid - free regime called the " edmonton protocol " have been made . the success rate in achieving insulin - independence in islet - transplanted patients has been greatly improved . however , the lifelong immunosuppressive regimes to prevent transplant rejection and recurrence of autoimmune responses to beta cells are expensive and decrease the quality of life . a variety of strategies to protect the transplanted islets from immune attack one of these involves preventing the activation of antigen - presenting cells . regulating the immune system by shifting the pathogenic effector cells to protective regulatory cells and blocking co - stimulatory pathways protective effects on transplanted islet cells can be gained by the transduction of genes for the expression of cytokines , antiapoptosis molecules , or growth factors . another strategy to protect islets from immune attack involves the microencapsulation of islets within synthetic polymers . there is easy passage of small molecules , such as glucose , amino acids , and insulin . oxygen deprivation presents another limit for the long - term survival of islets within a microcapsule . a major hurdle for islet transplantation is a lack of an islet source to meet the needs of all type 1 diabetic patients . embryonic , fetal , neonatal , or adult porcine islets are possible sources for transplantation , since easily available pigs have many physiological similarities to humans . however , there is a problem secondary to the hyperacute rejection of xenotransplants that carry the gal--gal antigen . another possible source of beta cells for transplantation is primary beta cells that are immortalized by transduction with oncogenes for expansion . these also have a conditional expression system that can reverse oncogene expression . however , this strategy carries the possibility of impaired insulin secretion and tumorigenicity . in particular , increasing the tolerance of islet transplants and reducing transplant rejection are necessary . there is also a need to find an unlimited source of insulin - producing cells that are required for the wider clinical application of islet transplantation . the engineered cell will become an alternative insulin - producing cell that will have an advantage over intact islets . non - beta cells may not be recognized by beta cell - specific autoimmune responses . a variety of cell types , including fibroblasts , hepatocytes , neuroendocrine cells , and muscle cells , have been engineered to produce insulin with varying degrees of success . since hepatocytes have a glucose - sensing system similar to that in pancreatic beta cells like glucose transporter 2 ( glut2 ) and glucokinase , they draw much attention as target cells for insulin production . a preclinical model of diabetes was corrected by autologous transplantation of primary hepatocytes that were non - virally transduced with a glucose - responsive promoter - regulated insulin gene construct . intestinal k cells , possible surrogate beta cells , contain the necessary enzymes for processing proinsulin to insulin and have exocytotic mechanisms . a murine enteroendocrine cell line expressing insulin under the control of a glucose - dependent insulinotropic polypeptide not only reversed diabetes when transplanted , but also produced insulin in response to glucose . transplantation of bone marrow mesenchymal stromal cells expressing insulin under the glucose - responsive early growth response gene ( egr-1 ) promoter resulted in the remission of diabetes in mice . the regulation of insulin production by glucose - responsive promoters in non - beta cells exhibits slow kinetics , which may cause hypoglycemia . this situation is due to the requirement for insulin transcription and translation , as compared with the rapid release of insulin from beta cells by exocytosis . pancreatic beta cells have unique characteristics specific to the production of insulin , such as specific peptidases , glucose - sensing systems , and secretory granules that can release insulin promptly by exocytosis in response to extracellular glucose levels . thus , it is very difficult to mimic this tight regulation of insulin production in response to physiological levels of glucose in non - beta cells . multiple studies have shown that it is possible to direct in vitro differentiation of stem and progenitor cells toward insulin - producing cells . embryonic stem ( es ) cells have the potential to generate unlimited quantities of insulin - producing cells . theoretically , es cells could be expanded indefinitely in the undifferentiated state and then differentiated into functional beta cells . two major strategies are used for the differentiation of es cells into insulin - producing cells : the embryoid body formation and the definitive endoderm formation . a recent report showed that the stepwise differentiation of es cells , which mimics endogeneous pancreatic development , could generate functional beta cells . however , the yield is still very low and the differentiated beta cells are much less functional as compared with primary islets . more efficient protocols need to be developed for the differentiation of es cells into mature functional beta cells . adult stem cells are the most important source for cell therapy for various disease models , as they are free from the ethical problems of es cells and could provide an unlimited resource . many studies reported that insulin - producing cells can be generated from adult stem / progenitor cells that are present in bone marrow , adipose tissue , liver , intestine , spleen , salivary glands , neuronal tissues , and umbilical cord blood . adult stem / progenitor cells from liver tissue are a good source for making insulin - producing cells , as the liver and pancreas share common bipotential precursor cells within the embryonic endoderm . isolated neurogenein 3 ( ngn3)-positive cells from the injured adult mouse pancreas or clonally identified cells from adult pancreatic islets and ductal populations have the ability to differentiate into cells with beta cell function . umbilical cord blood ( ucb)-derived mesenchymal stem cells ( mscs ) may be an ideal source of stem cells that can be obtained without pain or risk of viral contamination to the donor . ucb - derived mscs have a multi - lineage differentiation capacity under specific manipulation of the culture conditions . several studies have shown that ucb stem cells can be differentiated into insulin - producing cells . adipose tissue - derived stem cells ( adscs ) are located within the stromal vascular fraction of adipose tissue . adscs can be isolated in high numbers from human adipose tissue at low risk to the patient . an interesting study reported the successful differentiation of functional insulin - producing cells from the stem cells of human eyelid adipose tissue using a two - step culture condition . similar to other stem cells , pdx-1-transduced adscs derived from human or murine tissue could differentiate into insulin - expressing cells under specific culture conditions . thus , adscs have the potential as a useful source for cell replacement therapy in diabetes . despite the success of the differentiation protocols , as described in this review , none of the protocols are yet able to produce fully functional mature beta cells . further research is required to understand how endogenous beta cells differentiate and to develop methods for generation of sufficient functional beta cells for clinically applicable therapies for diabetes . approaches for in vivo regeneration are to stimulate replication of beta cells and induce neogenesis . this replication can be stimulated by pregnancy and diabetogenic stimuli , such as glucose and free fatty acids , suggesting that beta cell growth can be artificially induced . growth factors , such as activin a and hepatocyte growth factor , have been used to induce beta cell replication . other factors , like a combination of epidermal growth factor ( egf ) and gastrin , keratinocyte growth factor , betacellulin ( btc ) , and glucagon - like peptide-1 ( glp-1 ) or its long - lasting homolog , exendin-4 , have also been used to induce beta cell replication . in addition , members of the regenerating protein family , such as reg protein and islet neogenesis gene associated protein ( ingap ) , can stimulate proliferation of beta cells and have been investigated as potential therapies for diabetes . both glp-1 and egf have been combined with gastrin as a therapy to restore the beta cell mass . these treatments restored normoglycemia in autoimmune non - obese diabetic ( nod ) mice , both by restoring the beta cell mass and by downregulating the immune response . in a similar study , combination therapy with egf and gastrin induced neogenesis of human beta cells from pancreatic duct cells in vitro , as well as from human pancreatic cells implanted into immunodeficient nod . clinical trials are underway for beta cell regeneration in type 1 diabetes using synthetic exendin-4 ( ac2993 ) . further clinical trials using immune suppressors and egf and gastrin analogs ( e1-inf ) for type 1 and type 2 diabetes have been reported . in phase 2 clinical trials , ingap peptide revealed no consistent treatment effects on fasting glucose , insulin , or c - peptide in type 1 and type 2 diabetic patients , although the effects on hbalc and stimulated c - peptide were promising . the expression of beta cell transcription factors either with or without a signaling molecule , such as a growth factor , has shown to be a successful method of generating new insulin - producing cells . delivery of the pancreatic and duodenal homeobox-1 ( pdx-1 ) gene into the mouse pancreas or intraperitoneal injection of pdx-1 protein induced beta cell neogenesis and ductal proliferation . delivery of the pdx-1 gene along with the btc gene into the pancreas of streptozotocin - induced diabetic rats via ultrasound - targeted microbubble destruction normalized blood insulin and c - peptide levels . intraperitoneal injection of recombinant pdx-1 into streptozotocin - induced diabetic mice increased islet cell numbers and proliferation in pancreata . an exciting report demonstrated that expressing a specific combination of three transcription factors , pdx-1 , ngn3 , and musculoaponeurotic fibrosarcoma oncogene homolog a ( mafa ) , resulted in amelioration of hyperglycemia . the transcription factors were administered by an adenoviral mediated delivery reprogrammed pancreatic exocrine acinar cells into beta cells . these beta cells were indistinguishable from endogenous islet beta cells in size , shape , and ultrastructure . transcription factors , such as pdx-1 and neurod , have been used not only to stimulate new insulin - producing cells in the pancreas , but also to confer beta cell - like characteristics to non - islet tissue , such as in the liver and the intestine . systemic injection of recombinant pdx-1 resulted in expression of insulin and other genes related to pancreatic function not only in the pancreas , but also in the liver . delivery of the pdx-1 gene or neurod and btc genes to the liver resulted in the expression of insulin and other islet - specific genes in liver . these genes ameliorated hyperglycemia in diabetic mice . systemic delivery of pdx-1 carrying the vp16 transcriptional activation domain ( pdx-1/vp16 ) or ngn-3 and btc gene resulted in insulin production in liver . in vivo regeneration in the intestine has also been investigated . forced expression of pdx-1 , mafa , or glp-1 in intestinal epithelia by adenovirus - mediated gene transfer induced the expression of insulin and lowered blood glucose levels in streptozotocin - induced diabetic animals . the injection of stem / progenitor cells was shown to induce insulin - producing cells . it is unclear whether the injected stem / progenitor cells differentiate into insulin - producing cells or exert other effects that preserve or increase the beta cell mass . the injection of allogeneic splenocytes , in combination with complete freund 's adjuvant ( to prevent anti - islet autoimmunity ) , corrected diabetes in diabetic nod mice . however , it remains to be determined whether the injected splenocytes are the true source of insulin - producing cells . a successful clinical trial involving autologous hematopoietic stem cell transplantation and immune suppressor in diabetic patients although beta cell function was shown to be increased , the mechanism of action was unclear . in vivo islet cell regeneration therapy is very challenging . most methods are still in the early stages and are not yet ready for clinical application . islet transplantation is a promising strategy for the reconstitution of a functional beta cell mass . this method provides stable glycemic control without hypoglycemic episodes , as well as independence from exogeneous insulin . however , a shortage of islet donors is a major limiting factor for cell replacement therapy in type 1 diabetes . the differentiation of pluripotent stem / progenitor cells into insulin - producing cells creates a possible source for the generation of therapeutic insulin - producing cells . beta cell growth and differentiation factors , the expression of beta cell transcription factors , and the injection of stem / progenitor cells has been used to regenerate beta cells in vivo . most results have been obtained from studies with animal models , but very little has been tried clinically . the underlying mechanisms for the normal renewal process in adults will accelerate the clinical application of islet cell replacement and regeneration therapy for diabetic patients .

there are several possible reasons that can cause splenomegaly , for example , infectious mononucleosis , subacute infective endocarditis , miliary tuberculosis , gibraltar disease malaria , schistosomiasis , and kala - azar . many approaches have been applied to treat splenomegaly , for example , surgical splenectomy , splenic embolization , banding or ligation of the splenic artery and placing a percutaneous stent to the splenic artery . unfortunately , the current management of splenomegaly has intrinsic limitations and disadvantages : surgical splenectomy can relieve hypersplenism but the morbidity after splenectomy is high , some cases of hypersplenism are not significantly improved following banding or ligation of the splenic artery , splenic embolization is also used to deal with splenomegaly but complications such as portal thrombosis , sepsis and the essential exposure to ray limit its utility . in our clinical practice , we have initiated a new approach , that of total splenic vessel ( artery and vein ) ligations ( tsvls ) in paediatric patients with splenomegaly and our preliminary outcomes indicate the feasibility and safety of the approach . here the purpose of this paper is to evaluate the results obtained with the procedure of tsvls for paediatric patients . according to the health insurance portability and accountability act ( hippa ) guidelines , the protocol was approved by the institutional review board and all patients provided written informed consent . between june 2011 and july 2013 , 17 paediatric patients with splenomegaly caused by hereditary spherocytosis ( n = 3 ) , portal hypertension ( n = 12 ) , paroxysmal nocturnal haemoglobinuria ( n = 1 ) and thalassemia ( n = 1 ) , referred for treatment with tsvl , were screened for enrolment into this retrospective analysis . the diagnosis of splenomegaly was established by inspection of medical history , laboratory examination , ultrasonography and computed tomography ( ct ) examinations . the eligibility criteria for tsvl included : ( 1 ) thrombocytopenia ( platelet count of b100,000/mm ) , ( 2 ) the diagnosis of primary disease was unequivocal , ( 3 ) splenomegaly class ii or iii . patients with other severe disease ( such as liver failure , severe infection ) were excluded . all ligation procedures were performed by two experienced interventional radiologists who were experienced in conventional laparoscopic surgery . the patient was placed in a supine position and the left upper abdominal region was raised slightly . a laparoscope ( storse ) was placed at the umbilicus and the other three ports were inserted . after opening the gastrocolic ligament and turning up the stomach to expose the pancreatic edge , we identified and dissociated the splenic vessel ( one artery and two to three veins , commonly ) . then , we separated the distal and proximal parts of the splenic vein , and used clips to ligate the vein distally and proximally . postoperatively , nearly 90% of the spleen appeared dusky while the remaining 10% of the spleen remained normal , which was an attribute of the patency of the collateral arteries . peripheral blood cell parameters , including white blood cell ( wbc ) count and platelet ( plt ) count as well as red blood cell ( rbc ) count , were measured sequentially 3 days before and 3 days , 2 weeks after tsvl , and subsequently at dates with 3-month intervals during the 1-year follow - up period . but ct might have radiation hazards , so ct was prudently performed and only applied in 1 week before and 6 month after the operation . according to the health insurance portability and accountability act ( hippa ) guidelines , the protocol was approved by the institutional review board and all patients provided written informed consent . between june 2011 and july 2013 , 17 paediatric patients with splenomegaly caused by hereditary spherocytosis ( n = 3 ) , portal hypertension ( n = 12 ) , paroxysmal nocturnal haemoglobinuria ( n = 1 ) and thalassemia ( n = 1 ) , referred for treatment with tsvl , were screened for enrolment into this retrospective analysis . the diagnosis of splenomegaly was established by inspection of medical history , laboratory examination , ultrasonography and computed tomography ( ct ) examinations . the eligibility criteria for tsvl included : ( 1 ) thrombocytopenia ( platelet count of b100,000/mm ) , ( 2 ) the diagnosis of primary disease was unequivocal , ( 3 ) splenomegaly class ii or iii . patients with other severe disease ( such as liver failure , severe infection ) were excluded . all ligation procedures were performed by two experienced interventional radiologists who were experienced in conventional laparoscopic surgery . the patient was placed in a supine position and the left upper abdominal region was raised slightly . a laparoscope ( storse ) was placed at the umbilicus and the other three ports were inserted . after opening the gastrocolic ligament and turning up the stomach to expose the pancreatic edge , we identified and dissociated the splenic vessel ( one artery and two to three veins , commonly ) . then , we separated the distal and proximal parts of the splenic vein , and used clips to ligate the vein distally and proximally . postoperatively , nearly 90% of the spleen appeared dusky while the remaining 10% of the spleen remained normal , which was an attribute of the patency of the collateral arteries . peripheral blood cell parameters , including white blood cell ( wbc ) count and platelet ( plt ) count as well as red blood cell ( rbc ) count , were measured sequentially 3 days before and 3 days , 2 weeks after tsvl , and subsequently at dates with 3-month intervals during the 1-year follow - up period . but ct might have radiation hazards , so ct was prudently performed and only applied in 1 week before and 6 month after the operation . baseline demographic and clinical characteristics of patients as well as intraoperative and hospital characteristics are described in table 1 . the mean age of the 17 patients was 6.17 years , and the patients included 11 boys and 6 girls . there were 12 cases of portal hypertension , 3 cases of hereditary spherocytosis , 1 case of paroxysmal nocturnal haemoglobinuria ( pnh ) and 1 case of thalassemia . baseline demographic and clinical characteristics of patients all paediatric patients were successfully performed tsvl without the need for conversion to open operation or splenic embolization . the mean operative time was 50.9 min ( range : 32.1 - 70.3 ) and mean blood loss was 10 ml ( range : 5 - 20 ) . the mean postoperative hospital stay was 5.6 1.7 days ( range : 4 - 12 days ) . after operation 16 patients developed transient fever and were treated by intravenous fluids , and abdominal pain occurred in all 17 patients . pleural effusion was observed in three patients and those three patients were responsive to conservative management . no hernias were found at the port sites during our follow - up [ table 1 ] . the mean [ standard deviation ( sd ) ] splenic infarction rate of a total of 17 patients was 775 ( 5.1)% 6 months after operation . we next analysed our data in different aetiologies and we found the mean ( sd ) splenic infarction rate in patients with portal hypertension ; other aetiologies were 78.5 ( 5.4 ) % and 75.0 ( 3.2)% . there was no significant difference in the splenic infarction rate among the aetiological groups indicating that the effectiveness of tsvl may be independent of aetiologies [ figures 1 and 2 ] . splenic infarction rate of paediatric patients post - procedure ct scans with contrast ( a ) normal spleen ( b ) infarcted spleen figure 3 showed the chronological changes of peripheral blood count before tsvl and 3 days to 1 year after tsvl . after tsvl , the mean counts of plt and wbc increased significantly and reached the peak level on the second week and on 3 days after tsvl , respectively . then the counts of plt and wbc gradually fell , reaching a steady state in the third month . both the plt and wbc were significantly higher than pre - tsvl in the 1-year follow - up . as for the rbc counts , no significant chronological change was found either subsequent to tsvl or during the follow - up period . follow - up of ( a ) plt count , ( b ) wbc count , and ( c ) rbc count in paediatric patients subjected to total splenic vessel ligations . * significant change ( a ) , ( b ) and ( c ) at the time point in comparison to pre - operation all paediatric patients were successfully performed tsvl without the need for conversion to open operation or splenic embolization . the mean operative time was 50.9 min ( range : 32.1 - 70.3 ) and mean blood loss was 10 ml ( range : 5 - 20 ) . the mean postoperative hospital stay was 5.6 1.7 days ( range : 4 - 12 days ) . after operation 16 patients developed transient fever and were treated by intravenous fluids , and abdominal pain occurred in all 17 patients . pleural effusion was observed in three patients and those three patients were responsive to conservative management . no hernias were found at the port sites during our follow - up [ table 1 ] . the mean [ standard deviation ( sd ) ] splenic infarction rate of a total of 17 patients was 775 ( 5.1)% 6 months after operation . we next analysed our data in different aetiologies and we found the mean ( sd ) splenic infarction rate in patients with portal hypertension ; other aetiologies were 78.5 ( 5.4 ) % and 75.0 ( 3.2)% . there was no significant difference in the splenic infarction rate among the aetiological groups indicating that the effectiveness of tsvl may be independent of aetiologies [ figures 1 and 2 ] . splenic infarction rate of paediatric patients post - procedure ct scans with contrast ( a ) normal spleen ( b ) infarcted spleen figure 3 showed the chronological changes of peripheral blood count before tsvl and 3 days to 1 year after tsvl . after tsvl , the mean counts of plt and wbc increased significantly and reached the peak level on the second week and on 3 days after tsvl , respectively . then the counts of plt and wbc gradually fell , reaching a steady state in the third month . both the plt and wbc were significantly higher than pre - tsvl in the 1-year follow - up . as for the rbc counts , no significant chronological change was found either subsequent to tsvl or during the follow - up period . follow - up of ( a ) plt count , ( b ) wbc count , and ( c ) rbc count in paediatric patients subjected to total splenic vessel ligations . * significant change ( a ) , ( b ) and ( c ) at the time point in comparison to pre - operation the management of hypersplenism includes several possible approaches such as surgical splenectomy , splenic embolization , banding or ligation of the splenic artery and placing a percutaneous stent to the splenic artery . unfortunately , all those managements of splenomegaly have limitations and disadvantages , and we have not found the ideal method to deal with splenomegaly . although surgical splenectomy may effectively improve blood cell destruction caused by hypersplenism , the morbidity of severe complications after splenectomy is still high , ranging from 9.6% to 26.6% . in addition , splenectomy can affect the body 's immune function , increasing the risk of septic events . the effect of ligation of the splenic artery in some hypersplenism cases is not significant . percutaneous placement of a stent into the splenic artery seemed to be a promising technique in the treatment of splenomegaly and hypersplenism ; however , ensuring a sustained and long - term increase in plt and wbc counts was difficult as the splenic artery was incompletely occluded . splenic embolization was first performed by in 1979 , which might preserve spleen function but high complications associated with splenic embolization such as post - embolization syndrome , splenic abscess , splenic rupture , pneumonia , portal thrombosis and the essential exposure to ray limit its utility . here , we developed a new approach , total splenic vessel ligation ( tsvl ) , for splenomegaly . in our knowledge , this is the first study to evaluate the results obtained with tsvls for paediatric patients . previously , we only ligated the splenic artery but the effect was not significant , especially thrombocytopenia that was often recurrent in long - term follow - up , which was in accordance with the published article . so in 2011 , we started to ligate total splenic vessel including the splenic artery and vein . in this study , we retrospectively analysed our original experience of 17 paediatric patients who underwent tsvl . in 17 patients followed up for more than 1 year , we found that the mean splenic infarction rate of total 17 patients was 77.5% , and the counts of plt and wbc increased significantly , indicating that the effectiveness of tsvls was satisfactory and favourable . on the other hand , we found the mean splenic infarction rate in patients with portal hypertension and other aetiologies to be 78.5% and 75.0% , respectively . there was no significant difference in the splenic infarction rate among the aetiological groups indicating the effectiveness of tsvl may be useful for splenomegaly independent of aetiology . in this study , tsvl was technically successful in all paediatric patients , with no operation - related complication . although transient post - tsvl occurred in most paediatric patients , no death or other major complication was observed . these results indicate that tsvl is a safe approach in the treatment of paediatric patients with hypersplenism . as tsvl was performed by laparoscopic operation , the potential advantages of this approach are associated with improved cosmetic outcomes , incisional hernias , less risks for haemorrhage , and fewer wound complications , organ injuries , and less postoperative incisional pain . first , the sample size of the study included in the analysis was small and no control group was included ; the power to detect the outcomes was limited . randomized controlled trial comparing tsvl and other approaches for splenomegaly are needed in order to determine the risk of complications involved in the treatment of splenomegaly and long - term clinical efficacy . second , the follow - up time in our study was not very long ; so a longer follow - up was needed . last , the potentially existent significant risk of measurement bias might have more or less affected the accuracy of the results . first , the sample size of the study included in the analysis was small and no control group was included ; the power to detect the outcomes was limited . randomized controlled trial comparing tsvl and other approaches for splenomegaly are needed in order to determine the risk of complications involved in the treatment of splenomegaly and long - term clinical efficacy . second , the follow - up time in our study was not very long ; so a longer follow - up was needed . last , the potentially existent significant risk of measurement bias might have more or less affected the accuracy of the results . although our study including several demerits , further expanded follow - up studies are needed . based on the evidence , we make cautious conclusions that tsvls are safe and effective in the treatment of paediatric patients with splenomegaly , achieving a satisfactory long - term haematological response and benefit . tsvls may be an important factor for reducing the risk of major complications such as splenic rupture , pneumonia , and refractory ascites , and may serve as an alternative to surgical splenectomy or splenic embolization .

professional cleaning is a basic service occupation worldwide , and cleaning products are used daily in different environments , both indoors and outdoors.1,2 in recent years , a growing number of scientific studies have shown an association of cleaning work with respiratory adverse effects including asthma.35 in addition , skin diseases such as dermatitis of the hand have also been reported.68 one explanation for the observed respiratory adverse health effects among cleaning workers is chemical exposures deriving from cleaning products.2,911 several studies have investigated the relationship between adverse health effects , cleaning activity , and cleaning products.1219 several risk factors were identified including exposure to chemical substances via application of cleaning products and other cleaning activities . researchers have called for objective and more accurate estimates of occupational exposure to cleaning products in order to better understand their adverse effects.12 one major difficulty in this context is the multitude of cleaning products used , and the large number of chemical substances present in these products . moreover , safety data sheets ( sdss ) for professional cleaning products are made available to provide workers with health hazard information regarding substances or mixtures . the current eu classification system ( directives 1999/45/ec and 67/548/eec ) defines substances and preparations as dangerous if they are explosive ( e ) , oxidizing ( o ) , extremely or highly flammable ( f+ , f ) , very toxic ( t+ ) , toxic ( t ) , harmful ( xn ) , corrosive ( c ) , irritant ( xi ) , sensitizing ( xn or xi ) , carcinogenic ( t , xn ) , mutagenic ( t , xn ) , toxic for reproduction ( t , xn ) , or dangerous for the environment ( n ) . these labels are accompanied by risk phrases ( r - phrases ) , and typical r - phrases used for cleaning products are listed in the methods section . we identified frequently used professional cleaning products in switzerland and through a systematic sds analysis of these products , hazardous ( c , xn , xi ) substances were identified and listed . we plan to use these results in a future exposure study to better characterize exposures to substances presenting a health hazard among professional cleaning workers . to select a representative group of frequently used cleaning products , we mailed a letter to cleaning companies located in the french- and german - speaking cantons of switzerland ( n = 1476 , fig . the letter mailed to cleaning services was not available in romansh and italian languages , thereby excluding cleaning companies in the romansh and italian cantons of switzerland . cleaning companies were asked to specify cleaning activity , company size , and cleaning products used . cleaning companies were identified from the federal office of statistics using the code for cleaning companies ( nomenclature gnrale des activits conomiques ( noga code ) ( 2008 ) ) . companies were grouped into small ( 549 employees ) , medium ( 50250 employees ) , and large ( 250 employees ) . technical terms ( both french and german ) used in the cleaning sector were retrieved from the training manual used for professional cleaners in switzerland.20 to process the large number of responses , we used the teleform software ( cardiff teleform , version 10.5.2 , san diego , usa ) . flow - chart of the decision process for including and excluding ( non - french- and non - german - speaking cantons , unknown addresses , or uncommon types of cleaning ) cleaning companies in the study . 1 number of cleaning services selected for the study . the table shows response rates by company size . the letter included a list of cleaning products ( n = 488 ) from four major companies that manufactured , produced , and/or supplied products in switzerland . this list of cleaning products by brand names was finalized after discussions with a professional cleaning association , a medium - sized cleaning company , and a training center for professional cleaners . the cleaning companies were asked to mark the cleaning products they used from the provided list , and in the case where the cleaning products they used were not listed , the company was asked to write down these names before mailing the responses back . an excel spreadsheet was generated from teleform and imported to stata ( stata 12 , stata corp lp , lakeway drive , usa ) . cleaning products marked as being used by at least 10 cleaning companies were included in the systematic sds analysis . selected products were grouped into 10 product categories : floor cleaners ( fcs ) , general purpose cleaners ( gpcs ) , polishing products ( pps ) , carpet cleaners ( ccs ) , scale removing products ( srps ) , bathroom cleaners ( bcs ) , glass cleaners ( gcs ) , disinfection products ( dps ) , kitchen cleaners ( kcs ) , and other surfaces cleaners ( oscs ) . a comprehensive table was created listing all substances mentioned in the sdss under section 3 . section 3 in the sds lists all the ingredients in a mixture ( chemical name , cas number , and concentrations ) that are classified as health hazards and are present above their cut - off / concentration limits . section 3 of sdss is titled composition / information on ingredients and provides details about hazardous substances in the mixtures . names , substance identifier ( cas number ) , concentration or concentration ranges , and classifications according to current danger letters and r - phrases ( directives 1999/45/ec and 67/548/eec ) as well as new hazard classes and statements ( regulation ( ec ) no . 1272/2008 ) are presented in the table.2123 this was possible because switzerland has from 1 december 2010 to 1 june 2017 to replace the current classification system ( directives 1999/45/ec and 67/548/eec ) with the new ( regulation ( ec ) no . 1272/2008 ) , meeting the requirements of the globally harmonized system of classification and labeling of chemicals ( ghs).24 therefore , both the current classification and the new ghs labeling were available for this study . the regulations ( directive 67/548/eec , directive 1999/45/ec , ec no . 1272/2008 ) define substance concentration restrictions regarding the listing of substances in this section.2123 table 1 includes also the types(s ) of cleaning products ( fc , gpc , pp , cc , srp , bc , gc , dp , kc , osc ) where the chemical substances were present . a literature search was performed in pubmed ( http://www.ncbi.nlm.nih.gov/pubmed/ , 15 october 2013 ) by searching for substance name+exposure and cas number+exposure. if available , up to three studies were chosen for each chemical substance that was present in at least two selected cleaning products . health aspects , dermal and respiratory exposure studies , occupational exposure studies , exposure assessment methods , cleaning , and cleaning products. na : not available ; fc : floor cleaner ; gpc : general purpose cleaner ; pp : polishing product ; cc : carpet cleaner ; srp : scale removing product ; bc : bathroom cleaner ; gc : glass cleaner ; dp : disinfection product ; kc : kitchen cleaner ; osc : other surfaces cleaner . studies about substances listed in table 1 , when substances where present in at least two cleaning products . fragrances sometimes do not meet the criteria to be listed in section 3 composition / information on ingredients of the sdss ( e.g. low concentration ) . regulatory information if they are subjected to other regulations such as substances depleting the ozone layer ( ( ec ) no . 2037/2000 , persistent organic pollutants ( ec ) no . 850/2004 , and export / import of dangerous substances ( ec ) no . 689/2008).2527 names of fragrances , preservatives , and other chemical substances listed under section 15 of sdss are reported in the results section . cleaning products containing at least one substance listed with corrosive , irritant , and harmful symbols under the current eu classification system were counted and expressed in percentage for each of the 10 product categories . r - phrases relevant in this study are harmful by inhalation ( r20 ) , are harmful in contact with skin ( r21 ) , are harmful if swallowed ( r22 ) , causes burns ( r34 ) , causes severe burns ( r35 ) , is irritating to eyes ( r36 ) , is irritating to respiratory system ( r37 ) , is irritating to skin ( r38 ) , has risk of serious damage to eyes ( r41 ) , may cause sensitization by skin contact ( r43 ) , has danger of serious damage to health by prolonged exposure ( r48 ) , has possible risk of impaired fertility ( r62 ) , has possible risk of harm to the unborn child ( r63 ) , is harmful : may cause lung damage if swallowed ( r65 ) , repeated exposure may cause skin dryness or cracking ( r66 ) , and vapors may cause drowsiness and dizziness ( r67).the fractions of cleaning products , with at least one substance listed with the r - phrases r20 , r21 , r22 , r34 , r35 , r36 , r37 , r38 , r41 , r43 , r48 , r62 , r63 , r65 , r66 , and r67 , were expressed in percentage . to select a representative group of frequently used cleaning products , we mailed a letter to cleaning companies located in the french- and german - speaking cantons of switzerland ( n = 1476 , fig . the letter mailed to cleaning services was not available in romansh and italian languages , thereby excluding cleaning companies in the romansh and italian cantons of switzerland . cleaning companies were asked to specify cleaning activity , company size , and cleaning products used . cleaning companies were identified from the federal office of statistics using the code for cleaning companies ( nomenclature gnrale des activits conomiques ( noga code ) ( 2008 ) ) . companies were grouped into small ( 549 employees ) , medium ( 50250 employees ) , and large ( 250 employees ) . technical terms ( both french and german ) used in the cleaning sector were retrieved from the training manual used for professional cleaners in switzerland.20 to process the large number of responses , we used the teleform software ( cardiff teleform , version 10.5.2 , san diego , usa ) . flow - chart of the decision process for including and excluding ( non - french- and non - german - speaking cantons , unknown addresses , or uncommon types of cleaning ) cleaning companies in the study . 1 number of cleaning services selected for the study . the table shows response rates by company size . the letter included a list of cleaning products ( n = 488 ) from four major companies that manufactured , produced , and/or supplied products in switzerland . this list of cleaning products by brand names was finalized after discussions with a professional cleaning association , a medium - sized cleaning company , and a training center for professional cleaners . the cleaning companies were asked to mark the cleaning products they used from the provided list , and in the case where the cleaning products they used were not listed , the company was asked to write down these names before mailing the responses back . an excel spreadsheet was generated from teleform and imported to stata ( stata 12 , stata corp lp , lakeway drive , usa ) . cleaning products marked as being used by at least 10 cleaning companies were included in the systematic sds analysis . safety data sheets for cleaning products were obtained from the companies ' web sites . if sdss were not available , products were excluded from the sds analysis . selected products were grouped into 10 product categories : floor cleaners ( fcs ) , general purpose cleaners ( gpcs ) , polishing products ( pps ) , carpet cleaners ( ccs ) , scale removing products ( srps ) , bathroom cleaners ( bcs ) , glass cleaners ( gcs ) , disinfection products ( dps ) , kitchen cleaners ( kcs ) , and other surfaces cleaners ( oscs ) . a comprehensive table was created listing all substances mentioned in the sdss under section 3 . section 3 in the sds lists all the ingredients in a mixture ( chemical name , cas number , and concentrations ) that are classified as health hazards and are present above their cut - off / concentration limits . section 3 of sdss is titled composition / information on ingredients and provides details about hazardous substances in the mixtures . names , substance identifier ( cas number ) , concentration or concentration ranges , and classifications according to current danger letters and r - phrases ( directives 1999/45/ec and 67/548/eec ) as well as new hazard classes and statements ( regulation ( ec ) no . 1272/2008 ) are presented in the table.2123 this was possible because switzerland has from 1 december 2010 to 1 june 2017 to replace the current classification system ( directives 1999/45/ec and 67/548/eec ) with the new ( regulation ( ec ) no . 1272/2008 ) , meeting the requirements of the globally harmonized system of classification and labeling of chemicals ( ghs).24 therefore , both the current classification and the new ghs labeling were available for this study . 1272/2008 ) define substance concentration restrictions regarding the listing of substances in this section.2123 table 1 includes also the types(s ) of cleaning products ( fc , gpc , pp , cc , srp , bc , gc , dp , kc , osc ) where the chemical substances were present . a literature search was performed in pubmed ( http://www.ncbi.nlm.nih.gov/pubmed/ , 15 october 2013 ) by searching for substance name+exposure and cas number+exposure. if available , up to three studies were chosen for each chemical substance that was present in at least two selected cleaning products . health aspects , dermal and respiratory exposure studies , occupational exposure studies , exposure assessment methods , ; fc : floor cleaner ; gpc : general purpose cleaner ; pp : polishing product ; cc : carpet cleaner ; srp : scale removing product ; bc : bathroom cleaner ; gc : glass cleaner ; dp : disinfection product ; kc : kitchen cleaner ; osc : other surfaces cleaner . studies about substances listed in table 1 , when substances where present in at least two cleaning products . fragrances sometimes do not meet the criteria to be listed in section 3 composition / information on ingredients of the sdss ( e.g. low concentration ) . regulatory information if they are subjected to other regulations such as substances depleting the ozone layer ( ( ec ) no . 2037/2000 , persistent organic pollutants ( ec ) no . 850/2004 , and export / import of dangerous substances ( ec ) no . 689/2008).2527 names of fragrances , preservatives , and other chemical substances listed under section 15 of sdss are reported in the results section . cleaning products containing at least one substance listed with corrosive , irritant , and harmful symbols under the current eu classification system were counted and expressed in percentage for each of the 10 product categories . r - phrases relevant in this study are harmful by inhalation ( r20 ) , are harmful in contact with skin ( r21 ) , are harmful if swallowed ( r22 ) , causes burns ( r34 ) , causes severe burns ( r35 ) , is irritating to eyes ( r36 ) , is irritating to respiratory system ( r37 ) , is irritating to skin ( r38 ) , has risk of serious damage to eyes ( r41 ) , may cause sensitization by skin contact ( r43 ) , has danger of serious damage to health by prolonged exposure ( r48 ) , has possible risk of impaired fertility ( r62 ) , has possible risk of harm to the unborn child ( r63 ) , is harmful : may cause lung damage if swallowed ( r65 ) , repeated exposure may cause skin dryness or cracking ( r66 ) , and vapors may cause drowsiness and dizziness ( r67).the fractions of cleaning products , with at least one substance listed with the r - phrases r20 , r21 , r22 , r34 , r35 , r36 , r37 , r38 , r41 , r43 , r48 , r62 , r63 , r65 , r66 , and r67 , were expressed in percentage . the response rate to the letter sent to cleaning companies was the highest ( 50% ) for large companies ( 250 employees ) , and lower for medium ( 24% ) and small ( 11% ) companies ( fig . 1 ) . based on company responses , respondent companies employed > 40 000 employees . a total of 116 products were selected for sds analysis and 11 products were excluded because of missing sdss . in the 105 remaining selected products , one cleaning product contained 3.5 ( 2.8 ) chemical substances listed in section 3 of the sdss . although the type of glycol ethers varied greatly across cleaning products , they were often ( 20% of the products ) present in both small and large amounts ( 0.150% in the products ) . most glycol ethers were found in pps ( 48% ) , srps ( 42% ) , gpcs ( 37% ) , and fcs ( 36% ) ; some ( 20% ) were found in dps and kcs , and few ( 1011% ) were found in gcs , bcs , and ccs . the choice of surfactants was diverse but were present in 19% of the products and their concentration ranges varied greatly ( 0.130% in the products ) . we particularly focused on ethanolamines , known for their sensitizing properties.28 three ethanolamines were identified : monoethanolamine , triethanolamine , and 2-diethylaminoethanol . the most frequently used was monoethanolamine , which was present in eight products ( n = 8) : five fcs , two gpcs , and one kc . in all , 16% of the products contained organic solvents and the concentration ranges varied enormously ( 0.175% ) making up 75% of one of the products ( pp ) . other typical ingredients , although in lower concentrations , accounted for 18% of our substance list ( table 1 ) : phosphates , salts , detergents , ph - stabilizers , acids , and bases . quats , a substance class known for sensitizing and allergic responses among cleaners , were found in two products in 310% concentrations.2,29 fragrances were commonly ( 27% of identified substances ) found in low concentrations ( 0.015% ) , except when they also acted as a solvent ( 30% ) . interestingly , up to 91% of the selected cleaning products contained at least one substance that was subject to other regulations and are listed under section 15 of sdss . in total , 26 substances were found under section 15 of the sds ( table 2 ) . in all , 11 substances listed in section 3 of sdss were neither classified with danger symbol letters and r - phrases nor with hazard classes and categories . the remaining 117 substances were classified with danger symbol letters and r - phrases as well as with hazard classes and categories . of these , 82 substances were listed in addition to hazard classifications and statements ( ghs ) . in all , 4 substances were listed in sdss of more than 10 products , 17 substances in sdss of 510 products , 38 in sdss of 24 products , and 69 were mentioned only once in the sdss of the 105 selected cleaning products . by product categories , usually less than 40% of cleaning products were labeled corrosive ( c ) in section 3 of sdss , with exception srps ( 78% , fig . 2 ) . in most product categories , more than 70% of the products were labeled irritant ( xi ) , except for pps ( 33% ) . more than 50% of the products were labeled harmful ( xn ) , except for product category ccs ( 31% ) . percentages of products by product categories containing at least one substance labeled as corrosive ( c ) , irritant ( xi ) , and harmful ( xn ) in section 3 of sdss . floor cleaner ( fc ) , general purpose cleaner ( gpc ) , polishing product ( pp ) , carpet cleaner ( cc ) , scale removing product ( srp ) , bathroom cleaner ( bc ) , glass cleaner ( gc ) , disinfection product ( dp ) , kitchen cleaner ( kc ) , and other surfaces cleaner ( osc ) . 3 ) : corrosive ( r34 , r35 ) , irritant ( r36 , r37 , r38 ) , harmful ( r20 , r21 , r22 ) , sensitizing ( r43 ) , and others ( r41 , r62 , r63 , r65 , r66 , r67 ) . figure 3 shows the percentages of products ( all categories ) that have been labeled with these r - phrases in section 3 of sdss . percentages of cleaning products that have been labeled with corrosive ( r34 , r35 ) , irritant ( r36 , r37 , r38 ) , harmful ( r20 , r21 , r22 ) , sensitizing ( r43 ) , and other ( r41 , r62 , r63 , r65 , r66 , r67 ) r - phrases in section 3 of safety data sheets ( sdss ) . frequently used professional cleaning products contain a multitude of chemical substances with known health effects . cleaners may therefore be exposed to mixtures of health hazardous substances during their cleaning activity . it is important to note that sdss do not list all chemical substances present in a product , as regulations define substances and concentrations that must be listed.21,23 depending on the characteristics of the substances ( e.g. persistence , bioaccumulation , and toxicity ) , the concentration levels requiring listing are 1 or 0.1%.30 sensitizers were listed as a cleaning product ingredient under section 15 in the sdss only if required by other regulations.2527 interestingly , several substances found under section 15 of sdss have been associated with sensitizing mechanisms and/or allergic reactions . in our study , we selected frequently used cleaning products known from cleaning companies with five or more employees . the cleaning products included the four most popular brands that , according to a professional association for cleaning companies in switzerland , account for > 50% of the swiss professional cleaning products market . as mentioned above , we estimated that our results include products used by about 50% of the swiss cleaning workforce . this is because the large cleaning companies reported to have high numbers of employees ( more than several thousand ) . most cleaning products identified in this study were sold by global companies that sell and distribute their products worldwide . the results of this study may hold true for other industrialized countries similar to switzerland , although the cleaning product might be given a different brand name . not only is there a great diversity of chemical substances within cleaning products but also numerous companies offer hundreds of different cleaning products , which makes the task of assessing chemical substances used in professional cleaning products complicated . indeed , responses showed cleaning companies using products from 36 different product companies , and some reported that they produced their own products . we believe our results provide important information regarding type of cleaning products used in this industry , and common chemical substance classes found in these products and their health hazards . this knowledge should help in monitoring professional cleaners and their exposures to cleaning products and substances with known health effects . in addition , not only cleaning workers or those who are cleaning are at risk of exposure but also persons in rooms that were recently cleaned can potentially be exposed.3133 the main challenges in conducting an occupational exposure assessment for professional cleaners are the great number of cleaning products available and the large number of substances in these products . for further investigation , we recommend to focus on the 21 substances found in 5 products ( table 1 ) . especially of interest are the recognized sensitizers monoethanolamine and glycol ethers , frequently found in cleaning products . substances found in professional cleaning products may likely also be ingredients in cleaning products sold to the general public ; however , we did not survey these products.28 this work contributes to the efforts to better understand possible exposures to chemicals during the use of professional cleaning products . we found that hazardous substances in cleaning products are in particular fragrances , glycol ethers , surfactants , solvents , and to a lesser extent phosphates , salts , detergents , ph - stabilizers , acids , and bases . cleaning workers who are handling these products are therefore a group at risk for several occupational exposures . section 15 in the sds should be consulted , as several substances involved in sensitizing mechanisms and/or allergic reactions were also listed here . especially glycol ethers and ethanolamines are frequently used in cleaning products , and could therefore be involved in the development of adverse health effects like irritant or sensitizer - induced asthma , which has been found to be elevated among professional cleaners . concerning asthma , the presence of different aldehydes as fragrances is also of special interest . besides some sensitizers like ethanolamines , mainly irritants were found , suggesting that pathologies of the skin and the respiratory tract may also occur without mechanisms of sensitization . a simultaneous exposure to several hazardous chemical substances could potentially be involved in these pathologies . as professional cleaners represent a large workforce , and cleaning products are widely used , including in private cleaning , it is of great environmental and public health importance to better understand the exposures that may be caused by the use of cleaning products . our list of substances provides important information about which chemicals and hazards are relevant for further investigations in this field , and we plan to use these results for field exposure studies .

acute decompensation of chronic heart failure ( hf ) is a life - threatening situation with a short - term mortality of approximately 30% . intravenous positive inotropic agents play an important role in the treatment of acute decompensation in patients with systolic hf . although traditional inotropic agents such as , -adrenergic agonists and phosphodiesterase inhibitors appear effective for short - term improvement of symptoms , previous studies demonstrated that these agents increase morbidity and mortality by elevating intracellular cyclic adenosine monophosphate ( camp ) and calcium ( ca ) levels . despite optimum traditional treatment strategies , frequent hospitalization due to acute levosimendan ( ls ) is a ca sensitizer for the treatment of decompensated hf that increases troponin - c sensitivity against cytoplasmic ca without any modification of intracellular ca density . ls s sensitizing effect is dependent on the ca concentration , thus its affinity to cardiac myofilaments increases during systole and improves cardiac contractility . during diastole , due to a decrease in the ca concentration ls improves cardiac contractility , increases cardiac output and stroke volume , and decreases pulmonary capillary wedge pressure , pulmonary artery pressure , and systemic and vascular resistance [ 79 ] . neurohormonal activation and proinflammatory cytokines play key roles in this process . in severe hf , despite optimum medical treatment , elevated plasma brain natriuretic peptide ( bnp ) , and interleukin levels were reported to be independent risk factors for both morbidity and mortality . although previous studies displayed beneficial effects of both single - dose and repeated ls treatment on hemodynamics , symptoms , and inflammatory markers in patients with acute decompensated hf , there was no consensus on the frequency of its use . no data in the current literature exist concerning optimal dosing of ls in patients with acute decompensated hf . in this study , we researched the impact of single - dose versus repeated ls infusion on cardiac performance , immune reaction , and neurohormonal activation in patients with acute decompensated hf . the present study included patients hospitalized at cukurova university faculty of medicine cardiology clinic upon diagnosis of acute decompensated hf , with an ejection fraction less than 35% , and who were severely symptomatic ( new york heart association [ nyha]-iii / iv ) although taking intravenous diuretic and vasodilator therapy . a total of 29 patients who met the inclusion criteria were included in the study and divided into 2 groups consecutively ( 1:1 ) . a single - dose ls was given to patients in the first group and repeated ls was given to patients in the second group . the patients clinical conditions and echocardiographic and laboratory findings were assessed at the start of the study and at 3 days and 6 months after treatment . the following patients were excluded from the study : patients younger than 18 years of age . patients with restrictive or hypertrophic cardiomyopathy . patients with ischemic chest pain . patients who died during initial hospitalization patients with continuous ventricular tachycardia ( vt ) or ventricular fibrillation in the course of hospitalization . patients with a heart rate greater than 120/min . patients with a systolic blood pressure in the decubitus position less than 85 mm hg patients with serious renal failure ( creatine level > 2.5 gr / dl ) and severe liver failure patients unable to provide informed consent . all subjects gave informed consent and the study was approved by the local ethics committee . ls infusion in addition to standardized treatment was performed with a loading dose of 6 g / kg for 10 min following a 24-h infusion of 0.1 g / kg / min . ls was readministered in the first and third months to patients in the repeated treatment group . clinical characteristics , echocardiographic measurements , and laboratory variables prior to treatment were evaluated after 1 dose and after 6 months . however , patients in group 1 were assessed at an outpatient clinic during follow - up without hemodynamic monitoring . the study end - points were changes in clinical , biochemical , and echocardiographic variables at 6 month in both groups . transthoracic echocardiographic examination was conducted on all patients using an acuson sequola c 256 model ( acuson corporation , mountain view , ca , usa ) and a transducer with 3.5-mhz frequency along with ecg monitoring . examination was performed by finding convenient echocardiographic windows when the patient was in the decubitus position or lying on their left side . left ventricular systolic and end - diastolic interior dimensions were measured from the obtained cross - sections and the left ventricular ejection fraction was calculated automatically . moreover , left atrial systolic and end - diastolic sizes as well as septum and posterior wall thicknesses were measured from the same window . according to the modified simpson method , left ventricular systolic and end - diastolic volumes and ejection fraction ( ef ) were calculated . doppler velocity curves were recorded at a horizontal sweep speed of 100 mm / s while the patient held their breath after exhalation . sample volume was placed between the tips of the mitral leaflets at apical 4 chambers and left ventricular filling variants were measured by pulse wave doppler echocardiography technique . based on the records obtained , left ventricle diastolic early ( a wave ) and late ( e wave ) peak velocities and mitral deceleration time ( dt ) were measured and the e / a ratio was calculated . isovolumetric relaxation time ( ivrt ) obtained by placing the sample volume of continuous wave ( cw ) doppler in the left ventricle outflow tract to simultaneously display the end of aortic ejection , and the onset of mitral inflow was measured as the interval in between . time elapsed from the point where the mitral a wave finished to a point where the mitral e wave started ( a ) , and left ventricle ejection time ( b ) was measured . difference between the 2 measurements ( ab ) was considered as the total of isovolumetric contract and ivrt . the myocardial performance index was then calculated using the ( ab)/b formula . during tissue doppler measurements , specific attention was paid to ensuring that the ultrasonographic beams were parallel to the lateral and septal mitral annulus . peak systolic mitral annuler ( sm ) , early diastolic ( em ) , and late diastolic ( am ) velocities were measured with the sample volume placed at the junction of the lv wall and the mitral annulus of the septal and lateral myocardial segments from the 4-chamber view . the sm velocity occurs during the ejection period , while the em and am velocities correspond to passive ventricular filling and atrial contraction . for assessment of left ventricular systolic function , sm velocity was used . additionally , transthoracic echocardiography was performed by a second observer for the same 5 patients after the first echocardiogram to assess the intra- and interobserver variability . blood samples were taken from all patients from the antecubital vein for the measurement of bnp , interleukin ( il)-1 , il-2 , il-6 , and tumour necrosis factor ( tnf)- before and after ls infusion and at 6-month follow - up . the samples were collected in tubes containing edta , then centrifuged at 1500 rpm for a period of 5 min and stored at 40c until needed . after dissolution of serum samples , bnp level was measured by use of the electrochemiluminescent immunoassay method using phoenix pharmaceuticals ( burlingame , ca , usa ) commercial kits , and il-1 , il-2 , il-6 , and tnf- levels measured using the biosource ( grand island , ny , usa ) commercial kit . subjects data prior to treatment and at 3 days and 6 months post - treatment were compared using wilcoxon s signed - rank test . non - parametric data are expressed as percentages . a p - value < 0.05 was considered to indicate statistical significance . the present study included patients hospitalized at cukurova university faculty of medicine cardiology clinic upon diagnosis of acute decompensated hf , with an ejection fraction less than 35% , and who were severely symptomatic ( new york heart association [ nyha]-iii / iv ) although taking intravenous diuretic and vasodilator therapy . a total of 29 patients who met the inclusion criteria were included in the study and divided into 2 groups consecutively ( 1:1 ) . a single - dose ls was given to patients in the first group and repeated ls was given to patients in the second group . the patients clinical conditions and echocardiographic and laboratory findings were assessed at the start of the study and at 3 days and 6 months after treatment . the following patients were excluded from the study : patients younger than 18 years of age . patients with restrictive or hypertrophic cardiomyopathy . patients with ischemic chest pain . patients who died during initial hospitalization patients with continuous ventricular tachycardia ( vt ) or ventricular fibrillation in the course of hospitalization . patients with a heart rate greater than 120/min . patients with a systolic blood pressure in the decubitus position less than 85 mm hg patients with serious renal failure ( creatine level > 2.5 gr / dl ) and severe liver failure patients unable to provide informed consent . all subjects gave informed consent and the study was approved by the local ethics committee . ls infusion in addition to standardized treatment was performed with a loading dose of 6 g / kg for 10 min following a 24-h infusion of 0.1 g / kg / min . ls was readministered in the first and third months to patients in the repeated treatment group . clinical characteristics , echocardiographic measurements , and laboratory variables prior to treatment were evaluated after 1 dose and after 6 months . however , patients in group 1 were assessed at an outpatient clinic during follow - up without hemodynamic monitoring . the study end - points were changes in clinical , biochemical , and echocardiographic variables at 6 month in both groups . transthoracic echocardiographic examination was conducted on all patients using an acuson sequola c 256 model ( acuson corporation , mountain view , ca , usa ) and a transducer with 3.5-mhz frequency along with ecg monitoring . examination was performed by finding convenient echocardiographic windows when the patient was in the decubitus position or lying on their left side . left ventricular systolic and end - diastolic interior dimensions were measured from the obtained cross - sections and the left ventricular ejection fraction was calculated automatically . moreover , left atrial systolic and end - diastolic sizes as well as septum and posterior wall thicknesses were measured from the same window . according to the modified simpson method , left ventricular systolic and end - diastolic volumes and ejection fraction ( ef ) were calculated . doppler velocity curves were recorded at a horizontal sweep speed of 100 mm / s while the patient held their breath after exhalation . sample volume was placed between the tips of the mitral leaflets at apical 4 chambers and left ventricular filling variants were measured by pulse wave doppler echocardiography technique . based on the records obtained , left ventricle diastolic early ( a wave ) and late ( e wave ) peak velocities and mitral deceleration time ( dt ) were measured and the e / a ratio was calculated . isovolumetric relaxation time ( ivrt ) obtained by placing the sample volume of continuous wave ( cw ) doppler in the left ventricle outflow tract to simultaneously display the end of aortic ejection , and the onset of mitral inflow was measured as the interval in between . time elapsed from the point where the mitral a wave finished to a point where the mitral e wave started ( a ) , and left ventricle ejection time ( b ) was measured . difference between the 2 measurements ( ab ) was considered as the total of isovolumetric contract and ivrt . the myocardial performance index was then calculated using the ( ab)/b formula . during tissue doppler measurements , specific attention was paid to ensuring that the ultrasonographic beams were parallel to the lateral and septal mitral annulus . peak systolic mitral annuler ( sm ) , early diastolic ( em ) , and late diastolic ( am ) velocities were measured with the sample volume placed at the junction of the lv wall and the mitral annulus of the septal and lateral myocardial segments from the 4-chamber view . the sm velocity occurs during the ejection period , while the em and am velocities correspond to passive ventricular filling and atrial contraction . for assessment of left ventricular systolic function , sm velocity was used . echocardiographic measurements were repeated in 5 subjects the next day by the same investigator . additionally , transthoracic echocardiography was performed by a second observer for the same 5 patients after the first echocardiogram to assess the intra- and interobserver variability . blood samples were taken from all patients from the antecubital vein for the measurement of bnp , interleukin ( il)-1 , il-2 , il-6 , and tumour necrosis factor ( tnf)- before and after ls infusion and at 6-month follow - up . the samples were collected in tubes containing edta , then centrifuged at 1500 rpm for a period of 5 min and stored at 40c until needed . after dissolution of serum samples , bnp level was measured by use of the electrochemiluminescent immunoassay method using phoenix pharmaceuticals ( burlingame , ca , usa ) commercial kits , and il-1 , il-2 , il-6 , and tnf- levels measured using the biosource ( grand island , ny , usa ) commercial kit . subjects data prior to treatment and at 3 days and 6 months post - treatment were compared using wilcoxon s signed - rank test . non - parametric data are expressed as percentages . a p - value < 0.05 was considered to indicate statistical significance . our study consisted of 29 patients divided into 2 groups ( mean age 60.27.4 years ; 20 males , 9 females ) with severe cardiac failure ( nyha - iii / iv ) who were administered a single dose or repeated dose of ls . functional capacities of patients prior to treatment according to nyha were similar in both groups ( 3.80.4 vs. 3.70.4 , p=0.5 ) . at the end of the follow - up period , a more robust recovery was observed in group 2 ( nyha 1.50.7 vs. 0.90.6 ; p=0.03 ; figure 1 ) . the beneficial effects of ls on left ventricular parameters such as systolic volume , ejection fraction , myocardial performance index , and sm was evident beginning on the third day after administration of the initial dose of ls ( table 2 ) . left ventricular systolic volume , ejection fraction , and myocardial performance index improvement continued to 6 months only in group 2 ( table 2 and figure 2 ) . in addition , left ventricular end - diastolic volume decreased during follow - up only in group 2 . the patients baseline bnp , il-1 , il-2 , il-6 , and tnf- concentrations were elevated in both groups ( table 3 ) . seventy - two hours after ls infusion , a marked decline was detected in bnp , il-6 , and tnf- concentrations . decreased levels of bnp , il-6 , and tnf- remained at 6 months only in group 2 . all patients tolerated the treatment and were able to complete the infusion without adverse events . during the 6-month follow - up , no atrial fibrillation , ventricular tachycardia , or fibrillation developed and no patient died . ls infusion was shortly interrupted 11 times ( 20% ) during infusion upon development of hypotension . this study showed that repeated ls treatment led to sustained improvement in functional status and left ventricular systolic functions at 6 months post - treatment compared to a single - dose infusion . additionally , the treatment decreased the elevated levels of inflammatory cytokines considered as predictors of unfavorable outcome and mortality . a more intense therapy with repeated ls infusion in acute decompensated hf appeared as a reasonable option , especially for patients who could not remain stable without intravenous treatment . hf is a growing problem , especially as the elderly population is increasing , and for individuals and the general public with respect to re - hospitalization and frequent , prolonged hospitalization . although highly effective treatments including new drug therapies and devices have emerged , episodes of decompensation that require hospitalization still occur frequently . the loss of cardiomyocytes through apoptosis and necrosis is an essential cellular pathway associated with excessive neurohormonal activation that leads to progression of lv remodeling is . elevated circulatory levels of proinflammatory cytokines ( tnf- , il-6 , and il-1 ) accelerate apoptosis in cardiac myocytes and endothelial cells by increasing oxidative stress and lead to suppression of myocardial contractility , all contributing to clinical and hemodynamic deterioration of hf . furthermore , despite optimum medical treatment , sustained high plasma levels of these factors are independent predictors of morbidity and mortality in patients with advanced hf . therefore , changes in bnp levels during treatment of decompensated chronic hf were strongly associated with early re - hospitalization and mortality . several studies suggested that ls leads to a marked decrease in bnp and proinflammatory levels and provides clinical improvement in these patients [ 2224 ] . ls differs from traditional inotropic agents that display their positive inotropic effects by increasing intracellular ca concentrations , which may cause cardiomyocyte loss via the activation of apoptosis and necrosis . ls has a dual mechanism of action : ca - sensitization through binding to troponin c in the myocardium , and the opening of atp - sensitive k channels in vascular smooth muscle . these mechanisms evoke positive inotropy and vasodilation , which may result in improved cardiac output without increasing myocardial oxygen demand and impairing diastolic relaxation . or1896 , an active metabolite of the drug , maintains the clinical response between 7 and 9 days after continuous intravenous infusion for 24 h . recovery based on echocardiographic , laboratory , and hemodynamic variables could be considered a result of a sustained decrease in mechanical overload . the positive effects of both single - dose and repeated ls treatment in acute decompensated hf on hemodynamics , symptoms , and survival have been shown in various studies [ 3235 ] . in a study by mavrogeni et al . , monthly administration of ls for 6 months decreased left ventricle systolic and end - diastolic volume indices ( 16% and 13% , respectively ) , and increased left ventricle ejection fraction by 27% . in an another study by parissis et al . , 5 doses of repeated 24-h ls infusion were given at 3-week intervals , and patients were assessed 1 month after completion of the last dose . a decrease in cardiac volume and cardiac wall tension , an increase in left ventricle ejection fraction , and recovery in cardiac performance similarly , in our study , the beneficial effects of ls on left ventricular parameters such as systolic volume , ejection fraction , myocardial performance index , and sm became clear as early as the third day after administration of the initial dose of ls in both groups . improvement in left ventricular systolic volume , ejection fraction , and myocardial performance index continued for 6 months only in group 2 . in addition , left ventricular end - diastolic volume , which was unchanged after the initial dose in both groups , decreased during follow - up only in group 2 . we speculate that delayed amelioration in diastolic measures could be explained by late onset of accumulated efficacy of ls on left ventricular geometry , which is in agreement with the results of a previous study . one of the most important indicators of improved contractile functions with ls infusion is the increase in exercise capacity . in a tissue doppler echocardiography study reported by kasikcioglu et al . there was a significant increase in the exercise capacity assessed by a 6-min walking test , similar to the increase of sm after a single dose of ls . most of the patients ( 70% ) allocated to repeated ls therapy were in nyha class iv prior to treatment in our study . at the end of follow - up , functional status however , a similar recovery was not found in patients who received a single dose of ls . cardioprotective effects and stronger protection against decompensation with ls could be the underlying mechanism of a more robust functional recovery with repeated dosing of ls . significant reductions in il-6 , bnp , and tnf- levels were detected after the initial ls infusion in our study , which was in agreement with the results of previous studies [ 2224 ] . the beneficial effects of ls on inflammatory cytokines disappeared at the end of 6 months in the single - dose group . however , the levels of these markers remained stable in the repeated ls group during follow - up . clinical recovery and improved left ventricular function could eliminate the chronic inflammation process in these patients . inconsistencies in il-1 and il-2 levels may be explained by the small number of patients and intervening conditions such as infections , and transient hepatic or renal deterioration . additionally , this was not a randomized and controlled study ; no comparison was made with placebo or dobutamine . patients in the single - dose group were not evaluated echocardiographically and biochemical measurements were not performed during the first and third months . thus , the effects of repeated ls on biochemical and echocardiographic measures could not be compared . finally , the small sample size in this study precludes any valid subgroup analysis , thus we did not identify any confounders that may have interacted with treatment assignment and thus affect the outcome . additionally , this was not a randomized and controlled study ; no comparison was made with placebo or dobutamine . patients in the single - dose group were not evaluated echocardiographically and biochemical measurements were not performed during the first and third months . thus , the effects of repeated ls on biochemical and echocardiographic measures could not be compared . finally , the small sample size in this study precludes any valid subgroup analysis , thus we did not identify any confounders that may have interacted with treatment assignment and thus affect the outcome . in this study we showed that repeated ls treatment in patients with severe cardiac failure when compared to single - dose administration of ls , caused regulation and recovery of clinical symptoms and findings , hemodynamics , and inflammatory variants . the results suggest that repeated ls application in advanced hf is advantageous when compared to single - dose ls administration .

celiac disease is an inflammatory disorder in which the small intestine is damaged as a result of an excessive autoimmune response to gluten . however , the overall morbidity and mortality is moderately elevated in comparison to the general population . this is caused by the development of a number of associated disorders such as autoimmune diseases ( hypothyroidism , diabetes , autoimmune hepatitis , alopecia ) , osteoporosis and inflammatory bowel disease [ 1 , 2 , 3 ] . one of the complications of celiac disease , with the largest implication for the patient 's survival , is the development of small bowel malignancies . most studies report an increased risk of non - hodgkin lymphoma in celiac disease , though the risk for the individual patient is still very low . some evidence suggests that a gluten - free diet may reduce lymphoma risk [ 4 , 5 ] , though two case - control studies that screened for silent , i.e. untreated , celiac disease in lymphoma patients reported a less clear clinical association than perhaps thought [ 6 , 7 ] . in addition , patients with celiac disease are at risk of developing a small bowel carcinoma [ 1 , 8 , 9 ] . small bowel carcinomas are often overlooked in part because of their low prevalence , but also because diagnostic techniques such as video capsule and enteroscopy have only been recently developed . most small bowel cancers are adenocarcinomas located between the duodenum and the ligament of treitz [ 10 , 11 ] . surgical resection is the key therapy to obtain a good survival , but despite these measures the prognosis is dismal , with a 5-year survival rate of approximately 28% . it is unclear though whether earlier detection of celiac disease prevents the development of small bowel carcinoma . in this case report we want to raise the clinicians awareness for this silent killer in celiac disease and present the pitfalls in the clinical recognition as well as diagnosis of a small bowel adenocarcinoma . a 69-year - old male was referred to our tertiary center for a second opinion for a gastric motility disorder . his medical history revealed a dermatitis herpetiformis diagnosed 15 years previously which responded to dapsone . in the two preceding years , he had been investigated in another center because of prolonged upper abdominal pain and vomiting . initially anemia ( hb 7.6 mmol / l ) attributed to a benign ventricular ulcer were diagnosed and treated . due to persisting complaints , additional investigations were performed . laboratory parameters were normal and a duodenoscopy , duodenal biopsies , an abdominal ultrasound , colonoscopy , an abdominal ct scan and a small bowel enteroclysis showed no abnormalities apart from a raised but decreasing esr ( from 76 to 59 mm / h ) and a diverticular colon . just prior to referral to our center , he developed severe vomiting and gastric retention . a few years earlier his bmi had decreased from obese to normal . since then , his weight had been stable over the last year . laboratory investigations showed a slightly raised esr ( 25 mm / h ) , increased alt ( 127 u / l ) and vitamin b12 deficiency . revision of the small bowel follow - through and ct scan did not show any abnormalities . duodenal histology showed partial villous atrophy ( marsh 3a ) , and celiac disease was diagnosed . the patient was referred to a dietician to start a gluten - free diet and vitamin b12 therapy was initiated . for almost half a year , the patient was free of complaints . new duodenal biopsies excluded refractory celiac disease . upon follow - up he suffered from involuntary weight loss . three months later he presented with a palpable mass in the abdomen and anemia ( hb 6.9 mmol / l ) . a video capsule followed by a single balloon enteroscopy revealed a large process at the duodenal - jejunal junction ( fig . the lesion appeared to be a poorly differentiated adenocarcinoma , partly of the signet cell type . a ct scan suggested mesenterial infiltration in close relation to the stomach and several enlarged lymph nodes . laparotomy demonstrated an unresectable process with a close relation to the gastric wall , left kidney , aorta and pancreas . in view of these findings and a second ct showed an increase of the tumor mass and lymph node enlargement within 4 weeks . this case report gives rise to three important key messages of which we should be more aware in patients with ( untreated ) celiac disease . the first message concerns the fact that all patients with dermatitis herpetiformis have a gluten - sensitive enteropathy and that they should be advised to follow a gluten - free diet [ 13 , 14 ] . we present a patient with long - existing dermatitis herpetiformis who developed a small bowel carcinoma 15 years after initial presentation . our patient was never advised to follow a gluten - free diet , and in retrospect this may have been a contributing factor to the increased risk of small bowel carcinoma . the risk in dermatitis herpetiformis of developing additional autoimmune diseases is comparable with that in celiac disease , though the risk for developing a lymphoma is increased [ 15 , 16 ] . especially patients with dermatitis herpetiformis and autoimmune thyroiditis have a stronger predisposition to develop malignancies . the second message underlines the importance of considering a small bowel adenocarcinoma in patients with ( untreated ) celiac disease who present with unexplained abdominal pain . however the problem is that patients often present with nonspecific complaints and that the awareness of the possibility of a small bowel adenocarcinoma is low because of the overall low incidence . in the spectrum of all possible malignancies , only 2.5% of cancers in the digestive tract arise in the small bowel , of which 3040% present as an adenocarcinoma . why the small intestine is relatively protected against adenocarcinoma in comparison to the colon is unknown . possible factors protecting the small bowel include a rapid turnover of the small intestine mucosal cells , a fast transit time and thus short exposure of the small bowel to food and possible carcinogens , a small bacterial load in the small intestine , a well - developed iga - mediated immune system and an increased ( protective ) alkaline content of the small bowel . more recently , sanders et al . demonstrated that the small bowel produces less endogenous reactive oxidative species than the colon , resulting in a decrease in oxidative stress . in ( untreated ) celiac disease the ongoing inflammatory reaction together with a more permeable mucosal membrane to carcinogens and bacteria and the inability of the damaged epithelial cells to produce antioxidant enzymes may promote a malignant transformation . treatment of a small bowel adenocarcinoma is difficult and only based on surgical resection , with a 5-year survival of 32.5% in case of resection . there are some retrospective studies that suggest a benefit in survival after chemotherapy in patients with advanced small bowel adenocarcinoma [ 20 , 21 ] , though until now there are no specific guidelines because of the lack of randomized clinical trials . as such , the diagnosis can easily be missed on conventional radiological investigations . in the present case , the radiologists had not been made aware of the suspicion of a small bowel carcinoma . as a consequence , during the small bowel enteroclysis the tube was positioned just distal from the ligament of treitz and thus distal from the malignant process . since small bowel carcinoma in celiac disease often presents in the proximal small bowel , a position proximal from the ligament of treitz is preferable . with knowledge of the diagnosis , a second revision of the small bowel enteroclysis , which had been performed 2 years earlier , demonstrated a subtle process close to the ligament of treitz ( fig . in conclusion , all patients with dermatitis herpetiformis should be regarded as celiac disease patients and a gluten - free diet should be advised . it is unclear though whether this will prevent the development of a small bowel malignancy . also , we should be aware of the high risk of a small bowel carcinoma in celiac disease patients although a malignancy of the small intestine is rare . unexplained upper abdominal pain , weight loss and anemia should lead to additional investigations to exclude a small bowel malignancy in these patients . at last when considering this malignancy , one should make the radiologist aware of this suspicion . as a result

in june 2005 the centers for disease control and prevention ( cdc ) and the march of dimes , in collaboration with 35 professional and governmental organizations , convened a 3-day summit to discuss an agenda for preconception care programs , research , and policy . the summit was the result of internal workgroup discussions at cdc and an initial meeting with external stakeholders in november 2004 . prior to the november meeting , subject matter experts in 19 programs from 8 centers throughout cdc conducted a detailed review of the relevant literature . the 3-day summit was divided evenly into two components : a presentation series from preconception care practitioners , followed by a select panel meeting . more than 60 presentations on preconception care research and programs were made and subsequently discussed by the select panel to generate recommendations for improving maternal and child health outcomes through better preconception care . the goals of the select panel discussions were to define current scientific knowledge , to identify best practices , and to highlight key issues needing further attention that could be used as the basis for formulating recommendations and action steps . the select panel consisted of nationally recognized experts from a variety of disciplines representing different perspectives on preconception care services designed to promote women 's health and reduce adverse pregnancy and perinatal outcomes . the panel included experts in the fields of obstetrics , family practice , pediatrics , public health , nursing , reproductive health , toxic exposures , and chronic and infectious disease . the initial conceptual framework for deliberations and discussions was based on the acronym act ( accessible health care , comprehensive care , and timely provision of care ) proposed by dr . it was then expanded to include six a s : access , availability , affordability , acceptability , accuracy , and appropriateness . five organizing themes ( clinical practice , social marketing and health promotion , public health and community , public policy and finance , data and research ) served as the basis for discussion . one key topic of debate was the potential conflict in characterizing preconception care as preparation for pregnancy , as opposed to the broader promotion of women 's health . some panelists believed strongly that the focus must be on both women and infant health outcomes . in moving forward , the panel was conscientious not to carve preconception care out separately from good routine primary care ; however , panel members also asserted that even though the focus should be on preconception care , providers should also recognize that the scope of care should include comprehensive women 's health services . as one panel member said , preconception care should be happening at every interaction with a woman or man of reproductive age . we re just trying to get providers to reframe their thinking so we re achieving preconception health . some , while agreeing , argued that the ultimate goal was to improve perinatal outcomes , making this a part of , but distinct from , all well - woman 's health care . discussions often centered around the concern that insufficient scientific evidence currently exists for many preconception care interventions , for the best methods of integrating them into primary care , and for effectively delivering interventions as a package or even if they work . in contrast , other members believed that existing research findings were sufficient , and that moving in the direction of translation and action should be the next step . this theme was captured in one member 's comment , research is important ; there 's no doubt about it . but instead of just creating more new knowledge , let 's ask ourselves : why is it that the knowledge we created before is n't getting translated into action ? ultimately , the panel 's recommendations reflected both areas where sufficient evidence exists and those where more research is needed . many panel members supported recommendations that would advocate provider assistance for a woman and her partner in developing a reproductive life plan and in communicating her / their intentions through the implementation of that plan . as one panel member described it , [ primary care providers can ] develop the ability to help a woman write her reproductive health plan and facilitate her ability to carry this out , including the upgrading of this plan throughout her reproductive years . some participants suggested that the recommendations should strongly encourage all providers not only to ask a woman about her reproductive intentions , but to ask in a manner that conveyed to the woman that her decisions should be based on her personal preferences . this would allow the provider to help her choose the best contraceptive for meeting her life goals and to provide appropriate preconception awareness information . panelists generally agreed that the recommendations should focus on feasible actions that could have the greatest positive impact on health outcomes . this resulted in much discussion of fundamental issues , including 1 ) who gets preconception care women at high risk for adverse pregnancy outcomes or all women ; 2 ) what would yield the greatest return on investment ( e.g. , focus on the preconception period or the interconception period ; focus on all women or high - risk women ) ; 3 ) whether interventions could be packaged and targeted to reach specific populations or address specific needs of women ; and 4 ) how finance case and services , as well as the potential cost effectiveness and cost benefit of the proposed interventions ? the participants agreed that it is important to recognize what preconception care is and what it is not . preconception care is not a single visit but a continuum of care designed to meet the needs of a woman throughout the various stages of her reproductive life . the goal of the preconception care process is to make sure that the woman is healthy as she attempts to become pregnant [ 16 ] , and to promote her health and the health of her children throughout her reproductive lifespan . based on the select panel deliberations and its subsequently published recommendations , preconception care is defined here as a set of interventions that aim to identify and modify biomedical , behavioral , and social risks to a woman 's health or pregnancy outcome through prevention and management , emphasizing those factors that must be acted on before conception or early in pregnancy to have maximal impact . thus , it is more than a single visit and less than all well - woman care . it includes care before a first pregnancy or between pregnancies ( commonly known as interconception care ) . preconception care and interventions are designed to reduce perinatal risk factors and , for optimal effectiveness , must be successfully implemented before the start of pregnancy . the components of preconception care , including an array of interventions to address medical , psychosocial , and environmental risks associated with childbearing , augment routine well - woman care . current guidelines for perinatal care ( jointly issued by the american academy of pediatrics and american college of obstetricians and gynecologists ) recommend that all health encounters during a woman 's reproductive years , particularly those that are a part of preconception care , should include counseling on appropriate medical care and behavior to optimize pregnancy outcomes [ 8 , 9 ] . there is scientific evidence that certain preconception interventions are effective , yet at present they are not routinely and systematically applied . the framework for the recommendations and action steps to improve preconception health is based on two well - established models for community health and behavior change , the ecological and the lifespan models . the ecological model for medical care is constructed with the woman being the central player , while recognizing the significant roles of the larger community , the providers , and the institutions in ensuring the best outcomes for her ( fig . each level has primary responsibility for different aspects of health and well - being ; however , all levels are integrated and each plays a supportive role in the success of the other levels and the system as a whole . if one level is not fully engaged , the system will weaken , and the result may be poor health for the individual woman . in many regards the community and institution levels overlap ; institutions operate within the community , just as the community determines what services the institutions provide . using this framework , the proposed recommendations outline actions and responsibilities at each level and demonstrate the role that all sectors have in improving preconception care and ultimately the health of women and their families . the recommendations and action steps described here aim to address needs and gaps at each level . this framework recognizes that health is determined by factors across the lifespan and is intergenerational . improving preconception health will require , first , that the woman herself is achieving optimal health throughout her life . with women living in a healthy state , healthy children are more likely to result . as one panel member said , we should build on the pediatric model of anticipatory guidance [ and ] have consistent messages and care for women through the lifespan that will address women 's and preconception health before , between and beyond . furthermore , good health in infancy is likely to carry through to childhood , adolescence , young adulthood and older adulthood so that healthy infants become healthy adults and achieve their reproductive goals . this lifespan framework extends beyond the individual to promote health in the family and community . thus , the recommendations and action steps both address health needs across the lifespan and recognize that the individual , family , community , and institutions each have roles in ensuring that all women have lifelong optimal health . maximizing the potential impact of preconception interventions will require integration across the various levels of care and areas of expertise [ 5 , 1113 ] . changes in individual behavior , clinical practice , community programs , and public policies are recommended . in the context of clinical care , much can be done by primary care providers , but evidence suggests that many women have two primary care providers nevertheless , bundling the various preconception interventions has the potential to improve perinatal outcomes and reduce costs associated with adverse outcomes . these recommendations call for more integrated delivery of health promotion and care services for women rather than for greater fragmentation of the health - care system . integration is critical to moving forward , in consideration of competing priorities and limited resources , integration may achieve health - care economies of scale that produce more efficient delivery of services and more effective results in improving preconception health . for example , primary care providers potentially could routinely and efficiently use the tools available to screen for smoking , alcohol abuse , genetic risks , and occupational hazards ; endocrinologists , geneticists , and nutritionists could become an integral part of a preconception care strategy . moreover , settings such as family planning or sexually transmitted disease clinics must become part of an integrated approach in order to reach many women at high risk for adverse pregnancy outcomes . new models of care and well - designed quality - improvement efforts could foster the integration of preconception interventions . at the same time , preconception care must be tailored to meet the needs of the individual woman . given that preconception care is a process , not a visit , some recommendations will be more relevant to women at specific stages in their lives or those with varying levels of health risks . health promotion efforts , risk screening , and interventions would be different for a young woman who has never experienced pregnancy than for a 35-year - old woman who has had three children . women with chronic diseases , prior pregnancy complications , or behavioral risk factors may need more intensive interventions [ 5 , 6 , 11 , 12 ] . at the clinical level , it is especially important that individual interventions be tailored to the specific needs of the woman . such variations in interventions also place constraints on how interventions can and should be bundled or prepackaged . this special issue of the maternal and child health journal , as well as the mmwr recommendations and reports , emphasizes components of preconception care supported by research and an evidence base that documents the efficacy of specific interventions . for example , that increasing pre - pregnancy intake of folic acid reduces neural tube defects , and controlling diabetes prior to pregnancy yields better outcomes for mothers and babies [ 14 , 15 ] . as with prenatal care or well - child care , however , evidence may not be forthcoming that one regimented process of preconception care significantly improves outcomes . rather , the process of providing individualized screening , health education , and necessary interventions that can yield better outcomes . finally , implementing these recommendations to improve preconception health will take time . diffusion of innovation theory demonstrates how concepts and best practices tend to be disseminated and adopted . the innovators who lead current efforts to advance preconception care and the early adopters who run model programs are making only the first steps toward larger societal change . whereas the select panel 's action steps were designed to be short - term efforts , better implementing professional standards of care , modifying physician behaviors , developing effective health promotion messages , and adjusting payment mechanisms all will take time . based on the june summit presentations and subsequent expert panel deliberations , 10 recommendations were developed ( table 1 ) . these recommendations were based on four overarching goals that were identified as critical to help women reach optimal health and realize their reproductive goals . these four broad goals are 1 ) to improve both men 's and women 's knowledge , attitudes , and behaviors related to preconception health ; 2 ) to ensure that all u.s . women of childbearing age receive preconception care services screening , health promotion , and interventions that will enable them to begin a pregnancy in optimal health ; 3 ) to reduce risks indicated by a prior adverse pregnancy outcome through interventions during the interconception ( inter - pregnancy ) period that can prevent or minimize health problems for a mother and her future children ; and 4 ) to reduce the health disparities in adverse pregnancy outcomes . each woman , man and couple should be encouraged to have a reproductive life plan.action steps develop , evaluate , and disseminate reproductive life planning tools for women and men in their childbearing years , respecting variations in age ; literacy , including health literacy ; and cultural / linguistic contexts conduct research leading to development , dissemination , and evaluation of individual health education materials for women and men regarding preconception risk factors , including materials related to biomedical , behavioral , and social risks known to affect pregnancy outcomesrecommendation 2 . consumer awareness . increase public awareness of the importance of preconception health behaviors and preconception care services by using information and tools appropriate across various ages ; literacy , including health literacy ; and cultural / linguistic contextsaction steps develop , evaluate , and disseminate age - appropriate educational curricula and modules for use in school health education programs integrate reproductive health messages into existing health promotion campaigns ( e.g. , campaigns to reduce obesity and smoking) conduct consumer - focused research necessary to develop messages and terms for promoting preconception health and reproductive awareness design and conduct social marketing campaigns necessary to develop messages for promoting preconception health knowledge and attitudes , and behaviors among men and women of childbearing age engage media partners to assist in depicting positive role models for lifestyles that promote reproductive health ( e.g. , delaying initiation of sexual activity , abstaining from unprotected sexual intercourse , and avoiding use of alcohol and drugs)recommendation 3 . preventive visits . as a part of primary care visits , provide risk assessment and educational and health promotion counseling to all women of childbearing age to reduce reproductive risks and improve pregnancy outcomesaction steps increase health provider ( including primary and specialty care providers ) awareness regarding the importance of addressing preconception health among all women of childbearing age develop and implement curricula on preconception care for use in clinical education at graduate , postgraduate , and continuing education levels consolidate and disseminate existing professional guidelines to develop a recommended screening and health promotion package develop , evaluate , and disseminate practical screening tools for primary care settings , with emphasis on the 10 areas for preconception risk assessment ( e.g. , reproductive history , genetic , environmental risk factors) develop , evaluate , and disseminate evidence - based models for integrating components of preconception care to facilitate delivery of and demand for prevention and intervention services apply quality improvement techniques ( e.g. , conduct rapid improvement cycles , establish benchmarks and brief provider training , use practice self - audits , and participate in quality improvement collaborative groups ) to improve provider knowledge and attitudes , and practices and to reduce missed opportunities for screening and health promotion use the federally funded collaboratives for community health centers and other fqhc to improve the quality of preconception risk assessment , health promotion , and interventions provided through primary care develop fiscal incentives for screening and health promotionrecommendation 4 . follow - up to preconception risk screening , focusing on high priority interventions ( i.e. , those with evidence of effectiveness and greatest potential impact)action steps increase health provider ( including primary and specialty care providers ) awareness concerning the importance of ongoing care for chronic conditions and intervention for identified risk factors develop and implement modules on preconception care for specific clinical conditions for use in clinical education at graduate , postgraduate , and continuing education levels consolidate and disseminate existing professional guidelines related to evidence - based interventions for conditions and risk factors disseminate existing evidence - based interventions that can be used in primary care settings ( e.g. , brief interventions for alcohol misuse and smoking) develop fiscal incentives ( e.g. , pay for performance ) for risk management , particularly in managed care settings apply quality improvement techniques and tools ( e.g. , conduct rapid improvement cycles , establish benchmarks , use practice self - audits , and participate in quality improvement collaborative groups)recommendation 5 . use the interconception period to provide additional intensive interventions to women who have had a previous pregnancy which ended in adverse outcome ( e.g. , infant death , fetal loss , birth defects , low birthweight or preterm birth)action steps monitor the percentage of women who complete postpartum visits ( e.g. using hedis measures for managed care plans and title v maternal child health block grant state measures ) , and use these data to identify communities of women at risk and opportunities to improve provider follow - up develop , evaluate , and replicate intensive evidence - based interconception care and care coordination models for women at high social and medical risk enhance the content of postpartum visits to promote interconception health use existing public health programs serving women in the postpartum period to provide or link to interventions ( e.g. , family planning , home visiting , and wic) encourage additional states to develop preconception health improvement projects with funds from the title v maternal child health block grant , prevention block grant , and similar public health programsrecommendation 6 . offer , as a component of maternity care , one prepregnancy visit for couples and individuals planning pregnancyaction steps modify third party payer rules to permit payment for one prepregnancy visit per pregnancy , including development of billing and payment mechanisms consolidate existing professional guidelines to develop the recommended content and approach for such a visit educate women and couples regarding the value and availability of prepregnancy planning visitsrecommendation 7 . increase public and private health insurance coverage among women with low incomes to improve access to preventive women 's health , preconception , and interconception careaction steps improve the design of family planning waivers by permitting states ( by federal waiver or by creating a new state option ) to offer interconception risk assessment , counseling , and interventions along with family planning services . such policy developments would create new opportunities to finance interconception care increase health coverage among women who have low incomes and are of childbearing age by using federal options and waivers under public and private health insurance systems and the state children 's health insurance program increase access to health - care services through policies and reimbursement levels for public and private health insurance systems to include a full range of clinicians who care for womenrecommendation 8 . integrate components of preconception health into existing local public health and related programs , including emphasis on interconception interventions for women with previous adverse outcomesaction steps use federal and state agency support to encourage more integrated preconception health practices in clinics and programs provide support for cdc programs to develop , evaluate , and disseminate integrated approaches to promote preconception health analyze and evaluate the preconception care activities used under the federal healthy start program and support replication projects convene or use local task forces , coalitions , or committees to discuss opportunities for promotion and prevention in preconception health at the community level develop and support public health practice collaborative groups to promote shared learning and dissemination of approaches for increasing preconception healthrecommendation 9 . increase the evidence base and promote the use of the evidence to improve to preconception healthaction steps prepare an updated evidence - based systematic review of all published reports on science , programs , and policy ( e.g. , through the agency for healthcare research and quality) encourage and support evaluation of model programs and projects , including integrated service delivery and community health promotion projects conduct quantitative and qualitative studies to advance knowledge of preconception risks and clinical and public health interventions , including knowledge of more integrated practice strategies and interconception approaches design and conduct analyses of cost - benefit and cost - effectiveness as part of the study of preconception interventions conduct health services research to explore barriers to evidence - based and guidelines - based practice conduct studies to examine the factors that results in variations in individual use of preconception care ( i.e. , barriers and motivators that affect health - care use) support activities to translate research into clinical practice and public health actionrecommendation 10 . maximize public health surveillance and related research mechanisms to monitor preconception healthaction steps apply public health surveillance strategies to monitor selected preconception health indicators ( e.g. , folic acid supplementation , smoking cessation , alcohol misuse , diabetes , and obesity) expand data systems and surveys ( e.g. , prams and nsfg ) to monitor individual experiences related to preconception care use geographic information system techniques to target preconception health programs and interventions to areas where high rates of poor health outcomes exist women of reproductive age and their infants use analytic tools ( e.g. , ppor ) to measure and monitor the proportion of risk attributable to the health of women before pregnancy include preconception , including interconception , health measures and population - based performance monitoring systems ( e.g. , in national and state title v programs) include a preconception measure in the healthy people 2020 objectives develop and implement indicator quality improvement measures for all aspects of preconception care . for example , use hedis measures to monitor the percentage of women who complete postpartum visits panel recommendations and actions for each of the 10 recommendations , the panelists identified specific actions . for each action step , those persons primarily responsible for implementation as well as those who had supportive roles were listed . the action steps were designed as feasible and practical activities that could be undertaken in the near future and could result in change in the next 2 to 5 years . these goals , recommendations , and action steps form a strategic plan for improving preconception health and pregnancy outcomes . 2 ) , building from the smaller attainable actions steps to fulfilling a recommendation , to attainment of the four goals , and finally to the combination of the goals to reach the pinnacle of the pyramid a vision for better women 's health and improved pregnancy outcomes . fig . 2preconception care pyramid preconception care pyramid the recommendations presented here focus on individual health knowledge and behavior , clinical care , public health programs , and health - care policies , improving preconception health will require the involvement of and changes in other sectors , including education , housing , urban planning , and environmental health . moving forward , these sectors should be included as part of the comprehensive solution to improving women 's health and , by extension , the health of families . in all of the action steps , women and their families have a critical role to play in engaging the system and developing the consumer demand and culture to normalize preconception care as part of women 's health promotion and routine care . the 10 recommendations outlined in table 1 address individual , provider , system , and research needs that mirror the different levels described in the ecological model . under each of the recommendations there , action steps are focused on the development , evaluation , and dissemination of tools to help women and their partners make decisions regarding their reproductive health across the lifespan . the action steps clearly highlight the need to ensure that these tools are age - appropriate and culturally relevant and cover both general health topics and specific risk behaviors . the action steps recognize the importance of being able to integrate preconception health - care messages with existing health promotion activities whenever possible . both recommendations 1 and 2 call for action to be taken at both the individual and community level so that societal norms shift toward supporting optimal preconception health behaviors . recommendations 3 , through 6 call for actions to improve health - care services and are particularly focused on changing provider knowledge , attitudes , and behaviors . because the existing knowledge base has not been widely disseminated , the action steps call for new and continuing education activities to help all providers improve their skills and coverage of preconception care services . the use of quality improvement tools and techniques are suggested as a means of changing professional practices . in the current market - driven environment , it is clear that incentives such as pay for performance and risk management activities also must be developed to help encourage the provision of these services . recommendations 5 and 6 particularly focus more on improving preconception care and health for specific groups of women . recommendation 5 focuses on interconception care and the opportunity for the prevention of adverse pregnancy outcomes among those who are at risk for or have previously had pregnancy complications . in addition to action steps aimed at modifying provider practices , others encourage public health programs to do more identification and follow - up of women at risk . recommendation 6 suggests pre - pregnancy checkups or visits for couples to focus on their health and risks when they are trying to conceive . the action steps include consumer education and consolidated professional guidelines , as well as better third - party health insurance coverage of such visits as a part of prenatal and maternity coverage . action steps for recommendation 7 call for increased health - care coverage among uninsured , low - income women , specifically through medicaid . currently , most low - income women do not qualify for medicaid unless they are pregnant ; unfortunately , this is often too late for some health promotion and therapeutic interventions to have maximum impact on the health of the woman and her child . recommendation 8 describes a series of action steps that public health and community programs can take to improve preconception health by increasing the access to and use of preconception care services . these steps include using publicly funded women 's and children 's health programs to promote preconception health , to screen for health risks , and to refer women at risk to appropriate clinicians . for example , preconception health promotion messages are complementary to the purposes of family planning and hiv / std clinics . however , the use of publicly funded women 's and children 's health programs for these purposes will require that federal and state public health agencies minimize the categorical restrictions that often prevent true integration at the community level . the second two action steps encourage the adaptation , implementation , and evaluation of community - based programs that provide preconception services . there are model programs currently being implemented that could be modified and used to meet the unique needs of specific populations . a key component of these actions is to engage the community to develop the best methods for development , implementation , and evaluation of local preconception care programs . without community support and designs that truly represent the needs of the communities , recommendations 9 and 10 include action steps focused on the continuous quality improvement and planning feedback loop that supports excellence in health - care systems . research and evaluation . currently , a need exists for updating the systematic reviews of existing literature on preconception care interventions as well as increasing the knowledge base through qualitative and quantitative research projects . economic analyses of preconception care interventions are not often conducted , and a great deal of work must be done to understand the impact of interventions . limitations exist in many of the interventions currently available ; and effective interventions have not been developed in some areas . meeting the need for research and evaluation is a key factor in the ability to continue moving the field forward . in addition to research activities , ongoing monitoring systems typically managed by public health agencies are critical to program planning , development , and evaluation . this recommendation includes the use of surveillance systems such as the pregnancy risk assessment and monitoring system ( prams ) and the health employee data and information set ( hedis ) measures . improving preconception health requires changes in the knowledge , attitudes , and behaviors of individuals , families , communities , and institutions ( e.g. , government , health - care settings ) . the purpose is to improve the health of each woman before any pregnancy and to thereby affect the future health of the woman , her child , and her family . through the cdc preconception health initiative and the deliberations of the select panel on preconception care , a set of goals , recommendations , and specific actions steps have been developed to form a strategic plan . this work also recognizes the unique contributions and challenges faced by individual women and their families , communities , and institutions . as one select panel member put it , if you raise the level of health of women in a society , you ve raised the level of health of families and the community at the same time . the successful implementation of the recommendations will help achieve the vision for preconception health and pregnancy outcomes where all women and men of childbearing age have high reproductive awareness ( e.g. , understand risk factors related to childbearing).all women have a reproductive life plan ( e.g. , whether or when they wish to have children , how they will maintain their reproductive health).all pregnancies are intended and planned.all women of childbearing age have health - care coverage.all women of childbearing age are screened prior to pregnancy for risks related to the outcomes of pregnancy.women with a prior pregnancy loss ( e.g. , infant death due to very low birthweight or preterm birth ) have access to intensive interconception care aimed at reducing their risks . all women and men of childbearing age have high reproductive awareness ( e.g. , understand risk factors related to childbearing ) . all women have a reproductive life plan ( e.g. , whether or when they wish to have children , how they will maintain their reproductive health ) . all women of childbearing age are screened prior to pregnancy for risks related to the outcomes of pregnancy . women with a prior pregnancy loss ( e.g. , infant death due to very low birthweight or preterm birth ) have access to intensive interconception care aimed at reducing their risks .

radiotherapy represents a cornerstone of the treatment of breast cancer . in the past , the less advanced technology was the cause of severe damage to surrounding tissues in the breast , lung and chest wall . today the planning of irradiation fields guarantees the near total absence of collateral effects , in particular pneumonia and fibrosis induced by radiotherapy . radiation - induced pneumonia occurs classically 4 to 12 weeks after the radiotherapy and is characterized by fever , dry cough , dyspnea and alveolar opacities confined to the treatment port . however , bilateral lymphocytic alveolitis develops in both lungs after strictly unilateral breast irradiation and it presents with the histological , radiological and clinical pattern of bronchiolitis obliterans organizing pneumonia ( boop ) , now currently defined as cryptogenic organizing pneumonia ( cop ) . cop is a distinct clinico - pathological entity in the wide family of pulmonary fibrosis , and is characterized by patchy and often migratory peripheral air space infiltrates on chest radiography . cop has been associated with radiotherapy for breast cancer in some case reports , and it has been defined as " radiation - primed cop " . corticosteroid treatment in cop results in rapid clinical improvement of clinical symptoms and clearing of the opacities on chest imaging without sequelae . although the efficiency of steroids in cop has long been established , the precise dose , the duration of treatment , the use of boluses and the withdrawal have not been defined in radiation - primed cop . we report a case of cop after radiotherapy for breast cancer , presenting with fever , dyspnea , respiratory failure and bilateral pneumonia . the disease was successfully treated with systemic steroids but relapsed after the steroid withdrawal , and was definitively resolved after a second steroid course . this case emphasizes the importance of taking into account the possible occurrence of radiation - primed cop , and clarifies why only few women become affected with this disease after radiotherapy , what the optimal dose and duration of treatment should be , and if ( and , if so , which ) respiratory function tests can predict this complication . a 62-year - old woman was admitted to our out - patient clinic with a 2-week history of fever , dyspnea and dry cough . her chest radiography revealed pneumonia in the left lower lobe , already treated with antibiotics without benefit . she had undergone left mastectomy 1 year earlier , and subsequent adjuvant radiation treatment after the surgery for 50 days . in addition , immediately after the surgical procedure she had been given therapy with anastrozole ( arimidex ) , that was still in course at the onset of symptoms . computerized tomography ( ct ) scan showed a pulmonary infiltration with ground glass opacities in the left lower lobe outside the irradiation fields , diagnosed as pneumonia ( figure 1 ) . biochemical analysis revealed no abnormalities , except for elevated levels of c - reactive protein ( crp ) ( 18 mg / dl ) , and the arterial blood gas analysis showed a mild hypoxemia ( pao2 68 mm hg , paco2 31 mm hg , ph 7.43 , fio2 21% ) . the initial treatment was based on antibiotics for the clinical diagnosis of pneumonia , but clinical symptoms of dry cough and dyspnea persisted . given the ineffectiveness of prolonged antibiotic therapy , a fiberoptic bronchoscopy was performed with bronchoalveolar lavage ( bal ) and trans - bronchial biopsy ( tbbx ) . the bal fluid revealed a mild lymphocytic alveolitis ( total cell count : 840/l ; lymphocytes : 15% ; macrophages 84% ; neutrophils 1% ; cd4+/cd8 + t - lymphocyte ratio : 1.1 ) . tbbx was not diagnostic , and no malignant cells or pathogenic bacteria were discovered in the bal fluid . pulmonary function tests showed a mild restrictive defect with reduction of co - transfer : forced vital capacity ( fvc ) 82% of predicted value ( pred . ) , forced expiratory volume in 1 sec ( fev1 ) 78% pred . radiation - primed cop was diagnosed and a treatment with systemic steroids was started at the dose of 1 mg / kg of body weight with tapering to half dose every 4 weeks ( total duration of treatment 22 weeks ) . the ct scan , pulmonary function tests , blood gas analysis and crp at 12 weeks after the start of steroids were all normalized . four weeks after steroid withdrawal , the patient began to complain again of dry cough and dyspnea . a new ct scan revealed newly formed opacities in the upper right and in the middle lobes ( figure 2 ) . the patient underwent an identical treatment with systemic steroids , that resulted in an immediate improvement of symptoms , that persisted after 6 and 12 months . a complete normalization of ct images , pulmonary function tests ( fvc 94% pred . , kco 92% pred . ) , crp value ( 0.5 mg / dl ) and blood gas analysis ( pao2 72 mm hg , paco2 39 mm hg , ph 7.40 , fio2 21% ) was present at 12 months of follow up . reports of cop related to radiotherapy in breast cancer , in the form of case reports or literature analysis , date back to 15 years ago . cop is very similar to radiation pneumonia , but it involves lung segments that are far from the radiation field . patients who undergo radiation therapy to the breast may present alveolar lymphocytosis , even if they do not develop pneumonia . the negative effects of radiotherapy are well documented in the literature ( table 1 ) , but the histological and clinical pattern of radiation - primed cop in the lung seems to depict a distinct entity , with the epidemiological data , diagnostic and therapeutic algorithm not well defined yet . the prevalence of radiation - primed cop ranges between 2.5% and 1.8% [ 11 - 13 ] , but , to our knowledge , there are no italian or more local data available , that could be very useful to clarify the influence of differences in radiation method , dosing , and collimation on the occurrence of this pulmonary complication . synopsis of the literature on collateral effects of radiotherapy the primary role of pleural factors has been described in one sole report but not confirmed in an analysis of the recent literature . currently , an immunological mechanism is thought to be at the base of the disease development , as in hypersensitivity pneumonitis : irradiation provokes a tissue damage with sensitization of autoreactive lymphocytes , which react with pulmonary tissues . however , it is also possible that lymphocytes are not the key cells in the pathogenesis of radiation primed cop ; mast - cells could play a role , as in idiopathic cop and hypersensitivity pneumonia caused by inhaled antigens . in addition , some studies have shown that radiotherapy induces gene activation and transcription , cytokine release and fibroblasts activation . the cause of the migratory pattern and involvement of lung tissue away from the radiation fields remains unclear . therefore , the role of diagnostic methods like bronchoscopy with bal appears essential and well defined to identify the alveolar pattern of lung involvement . not equally defined is the role of tbbx , because a specific histological pattern has never been reported . from a clinical point of view , we limited the invasive procedures to tbbx due to the first result of absence of malignant cells or specific bacterial results , and the fast response to treatment . considering how many women undergo surgery and radiotherapy for breast cancer , radiation - primed cop is not so common , and probably different individual elements and/or factors related to the dose and length of radiation therapy a general review of the literature by cordier shows that the modifications of pulmonary function tests are generally mild or moderate , frequently consisting in a restrictive defect , even if an obstructive pattern can be present in patients with smoking habit or affected by chronic obstructive pulmonary disease . however , since the second half of the 1980s no report has clarified the changes of lung volume , blood gas analysis or co - transfer factor during cop , nor the possible risk patterns of lung damage detectable before the breast surgery and radiotherapy . . showed a relation between radiation - primed cop and hormone therapy with tamoxifen , anastrozole and toremifen , reporting a significant association ( or 3.12 , 95% ci 1.12 - 8.68 , p = 0.03 ) . our patient received anastrozole , but it was interrupted at the first occurrence of respiratory symptoms . however , it is rather difficult to precisely determine the influence of the withdrawal of this drug on the occurrence of the pulmonary complication , and alternatively on the outcome of the breast cancer . it seems really important to know which drugs represent a real risk factor for this complication , when they are able to exert a significant role and why this disease occurs in only few patients , with a reported greater incidence in subjects aged more than 50 years . in radiation - primed cop , as in other unknown diseases , therapy with systemic steroids is effective even if relapses are described after withdrawal . the doses of systemic steroids range from 0.5 to 1 mg / kg of body weight a day , but due to the serious unwanted effects of these drugs , like diabetes and osteoporosis , especially in elderly women , the literature is not updated about the lowest effective dose that can be used and the timing of dose tapering . this case report shows a postmenopausal woman submitted to surgery and radiotherapy and successively to hormone - therapy for breast cancer . the patient presented symptoms of fever , dry cough and dyspnea 4 months after radiotherapy ; radiological signs resembled pneumonia ; bal was suggestive of lymphocytic alveolitis ; all therapies were ineffective , but systemic steroids induced the complete resolution of lung opacities and symptoms . however , to date , radiation primed cop still remains unclear , with unknown causes , risk factors , role and timing of non invasive diagnostic tests , e.g. pulmonary function tests , and of invasive tests such as bronchoscopy with bal and tbbx , except for the possibility of excluding a diagnosis of cancer or tuberculosis . thus , we think it would be very useful to obtain epidemiological data about all women undergoing surgery and radiotherapy for breast cancer , to reveal possible relationships between radiation , hormonetherapy , and individual factors , in particular concerning the respiratory function before and during the treatment . none of the authors has any conflict of interest to declare in relation to the subject matter of this manuscript .

effects of endocrine disrupting chemicals ( edcs ) on human health and wildlife are receiving growing attention for the next generation 's health and have been known to interfere with endocrine systems by mimicking , blocking , and triggering actions of hormones and implicated with toxic effects , for example , disorders in development and reproduction . among edcs , bisphenol a [ bpa , 2,2-bis ( 4-hydroxyphenyl ) propane ] is widely used for a variety of applications , for example , baby feeding bottles , food - can lining , and sealants in dentistry . in addition , 4-tertiary - octylphenol ( op ) and 4-nonylphenol ( np ) of alkylphenols ( aps ) have been used to make alkylphenol ethoxylates , nonionic surfactants applied as emulsifying , wetting , or stabilizing agents in industries , and various consumer products including detergents and pesticide formulations . due to the wide uses of these phenols , it has been speculated that human exposures to environmental phenols may be widespread [ 36 ] . thus , concerns about various adverse health effects caused by edcs are increasing , and rigid risk assessment for edcs throughout valid biomonitoring studies has been called for . particularly , considering low - body weights and susceptibility , we suspect that body burden or real exposure level of infants or children to bpa or aps is expected to be heavier than those of adults . in the view of susceptibility , the exposures to environmental phenols in infants and children have got the public attention because edcs treat the second generation 's health , for example , genital malformations , testicular abnormalities , impairment in fertility or sexual functions , and neonates are considered to be a vulnerable subgroup to xenobiotics [ 7 , 8 ] . therefore , environmental phenols including bpa and aps should be continuously monitored for achievements of public heath , particularly for infants and children . contamination of ecds in colostrums raises concerns gravely because neonates , who are solely dependent on colostrums , are considered to be a high susceptible to edcs . detoxifying enzymes of neonates would not be fully developed at this early time point and exposure to edcs during the critical periods of developments could cause morphologic and functional alterations by influencing growth , reproduction , and development [ 7 , 8 ] . considering the characteristics of edcs that affect the second generation 's health , we need biomonitoring of edcs in colostrum , which is the main route of exposure to edcs for breast - fed infants . a number of investigations have reported the occurrence of several environmental chemicals such as persistent organic pollutants ( pop ) , polychlorinated dibenzo - dioxins ( pcdds ) , and organochlorines ( ocs ) in breast milk [ 911 ] . bpa , op , and np have the potency to partition into breast milk , since they are lipophilic compounds , which have octanol - water partition coefficient value ( log p or kow ) around 3 - 4 [ 12 , 13 ] . concerning the phenol exposure sources , we have studied various environmental sources ; however , we could not find crucial exposure sources , yet [ 14 , 15 ] . in a case of np , dairy products and sea food were suspected as its exposure sources [ 16 , 17 ] . thus , we focused on the consumption of dairy products and sea food or the use of cosmetics or surfactants to find phenol exposure routes in this study . in addition , we established a sensitive analytical method for bpa , op , and np in human colostrums and performed biomonitoring of these phenols among korean lactating women 's colostrums to assess risk of bpa and aps for breast - fed neonates . study subjects were 325 lactating mothers , who stayed in postpartum care centers in seoul , republic of korea . all subjects consented to participate in this study and donated their colostrums ( 10 ml ) . colostrums were collected in dark glass tubes with glass caps and stored 20c prior to analyses . we also obtained their questionnaires addressing physical characteristics , lifestyle patterns including dietary habits , and health and pregnancy - related properties [ 14 , 19 ] . all of the above procedures were approved by the institutional review boards of inje university , paek hospital ( seoul , republic of korea ) . throughout the entire procedure , we analyzed total forms ( conjugated and free phenols ) and free forms of bpa and aps for each colostrum sample with / without enzyme hydrolysis , respectively . the concentration of conjugated phenols was calculated after subtracting the amount of the free form of phenols from that of total phenols . to determine total forms of the three phenols levels in colostrum , we established an optimal method with modification of the bpa analysis . in brief , 100 ul of internal standard ( 125 ng / ml of bisphenol b ( bpb ) , tokyo kasei chemical , tokyo , japan ) , 120 ul of 2.0 m sodium acetate , and 48 ul of -glucuronidase ( 2,784 u ) ( sigma , st . the mixture was incubated at 37c for 5 hours ; 4 ml of 2-propanol was added and mixed thoroughly . thereafter , the mixture was centrifuged ( 3,000 g , 20 min ) , and 3 ml of supernatant was transferred to new glass tubes . this extraction was repeated , and total transferred 6 ml of supernatant was evaporated . after the evaporation , the residue was dissolved with 250 ul of 60% acetonitrile , and this solution was centrifuged ( 14,000 rpm , 10 min ) . thereafter , 200 ul of the supernatant was transferred to a vial , and 50 ul of the supernatant was injected for lc / ms / ms analysis . the lc / ms / ms system was composed with waters alliance 2695 xelc / ms / ms ( waters , watford , uk ) and zobax sb - c18 ( 5 m , 4.6 250 mm , agilent , usa ) . separation was accomplished with a gradient mode : mobile phase a , water : mobile phase b , acetonitrile : flow rate 0.3 ml / min , and ratio of a to b : 03 min , 70 : 30 ; 3 - 4 min , from 70 : 30 to 95 : 5 ; 46 min , from 95 : 5 to 100 : 0 ; 625 min , 100 : 0 ; 2530 min , from 100 : 0 to 70 : 30 . the waters alliance 2695 quattro premier xe was used in negative ion esi mode . the electrospray ionization ( esi ) settings were as follows : capillary voltage , 3.5 kv ; cone voltage , 40 v ; desolvation gas - flow ( argon gas ) , 800 l / hr ; cone gas - flow , 20 l / hr ; collision energy , 20 v. to determine concentrations of the free forms of phenols , we followed the procedures described above except enzyme hydrolysis . distribution normality of the phenols levels was tested with shapiro - wilk w test . if their probability ( w ) is < 0.05 , their distribution is considered not to follow normal distribution . nondetectable was assigned a value of half of the minimum value of detected each phenol level for further statistical analyses . considering each value 's characteristic ( normality and nominal , continuous , or ordered values ) , wilcoxon test , fisher 's exact test , spearman 's rho , and regression analysis were used for the study analyses . the statistical package of jmp version 4 ( sas institute , cary , nc , usa ) was used for all analyses . mothers were 30.67 3.45 years old , and , as expected , their body mass indices ( bmis ) were increased during the pregnancy ( median of before and after : 20.19 and 23.53 kg / m , resp . ) . based upon education level , occupation , and monthly household income , approximately , 11% ( n = 35 ) of the mother have clinical disease(s ) , such as toxemia ( n = 22 ) , thyroid disorders ( n = 6 ) , and gastritis ( n = 1 ) . similar number of the infants were sick at birth ( n = 34 ) , and most of the sick babies were recovered within 2 weeks after birth or on the therapy except two infants who were born with a congenital malformation . there was no association of disease presence between the mothers and their infants ( p = 0.66 ) . we established a sensitive analytical method to measure bpa and aps in colostrum samples with lc / ms / ms . limit of detection ( lod ) and limit of quantification ( loq ) were calculated with signal to noise ratio 10 and 30 , respectively . the calibration curve for simultaneous analyses of the three phenols was obtained within 5 different concentrations in phenols - spiked milk . the distributions of the three phenols were left skewed near to zero , that is , nondetectable values and did not follow normal distributions ( ps < 0.01 by shapiro - wilk w test for nonnormality ) . ranges of the three phenols ' levels in colostrum were shown in table 2 . op and np were not detected in most colostrum samples as free forms without any metabolism . in addition , there was a significant positive correlation between total op and np levels ( = 0.37 , p < 0.01 by regression analysis ) and somewhat positive association between total op and bpa levels ( = 0.06 , p = 0.09 ) . finally , we could estimate the daily exposure to the phenols in the infants via their mothers ' milk and determined that the current exposure levels to bpa and np were lower than their tolerable daily intakes ( tdis ) ( table 2 ) . to identify the potential routes of phenols exposure from environment , we investigated associations between phenol levels in colostrum and exposure sources . we observed that dairy products intake and detergents use were positively correlated with total bpa levels ( p < 0.05 ) ( table 3 ) . however , we did not find any statistically significant association between phenol levels and other exposure ( table 3 ) including air pollution at resident area or use of plastic wrap . considering no associations between mothers ' and infants ' disease presence mothers ' age was not associated any phenol levels ; however , embryonic period in neonates showed somewhat negative association with np levels ( = 0.25 , p = 0.09 ) . concerning phenols disposition at adipose sites , we studied effects of phenol exposure on bmi . as result , we found some positive associations between levels of mothers bmi ( before and after delivery ) and np ( = 0.09 , p = 0.01 and = 0.04 , p = 0.19 , resp . ) or op ( = 0.07 , p = 0.07 and = 0.03 , p = 0.34 , resp . ) . however , there were some negative associations between mothers bmi ( before and after delivery ) and levels of bpa ( = 0.11 , p = 0.07 and = 0.09 , p = 0.12 , resp . ) . secondly , we found that levels of total np in mothers or infants with diseases were higher than those in health subjects ( table 4 ) . in the case of mothers ' toxemia , a major mother disease ( n = 22 ) , we found more strong association between the disease and np exposure ( p < 0.01 ) . for neonates ' major disease , jaundice ( n = 13 including recovered cases ) , we could not find any significant association between the disease and the 3 phenol levels ( ps > 0.05 ) . in addition , neonates with congenital malformation ( n = 2 ) showed somewhat high levels of bpa , np , and op rather than others ( p = 0.07 , 0.11 , and 0.27 , resp . ) . in detail , the colostrum of the infant with congenital malformation of uvulas showed quite high levels of total np and op ( medians , 21.5 and 18.1 ng / ml , resp . ) compared to others ' levels ( medians < lod , i.e. , < 0.5 and < 0.3 ng / ml , resp . ) . considering physical complexity as a matrix of colostrum , proper preparations of colostrums specimens are required . recently , many researchers have used solid phase extraction ( spe ) for this purpose . considering broad use of bpa in plastic therefore , we performed liquid - liquid extraction ; even this preparation was time - consuming and labor intensive . in a case of biomonitoring environmental phenols , especially bpa , a general problem is background contamination during the analyses , which interfere with quantification at low concentrations of bpa . therefore , we used glassware throughout the entire analytical procedure in order to avoid possible contamination of bpa . blank tests , which were conducted with water instead of colostrum , were also performed at every daily experiment to confirm an absence of environmental phenol contamination in the whole of our experimental process . in addition , we tried to our best to avoid a drawback of lc / ms / ms analyses , for example , over estimation in low phenol - contained samples . thus , we established an optimal condition to measure bpa , op , and np in the colostrum samples . we also performed analyses of hplc / fld to confirm lc / ms / ms results in low phenols samples ( < medians of each phenol : n = 50 ) and obtained high reproducibility between two analyses ( cvs < 15% ) . among the present 325 colostrum samples , 70.6% of them have detectable levels of bpa in colostrum ( table 2 ) . mendonca et al . recently reported similar detection frequency of bpa in usa breast milk for 315 months infants , that is , 75% . considering the relatively high frequency of bpa detection in colostrum samples , we suggest a high potential for exposure of breast - fed infants to bpa via colostrum . in addition , median level of total bpa ( 7.8 ng / ml ) in the present colostrum study is somewhat higher than those in other biomonitoring studies with breast milk samples [ 12 , 23 , 24 ] . for example , another study with 101 colostrum samples detected bpa at a range of 17 ng / ml and a mean level of 3.41 ng / ml . however , they used convenient elisa methods and detected bpa in all samples without confirmation of bpa free system for analyses , for example , blank tests . mendonca et al . also reported quite low levels of total bpa ( median , 0.8 ng / ml ) in small number of infant breast milk samples ( n = 23 ) . on the basis of three phenol levels in colostrum , volume of daily intakes of milk ( 500 ml ) , and mean body weight of the baby subjects ( 3.24 0.46 kg ) , we estimated the daily exposure of infants to bpa , op , and np ( table 3 ) . the estimated exposure levels of bpa and np via colostrum in korean neonates appear to be safe , compared to their tdis [ 3 , 8 ] . in a case of op , we can not determine whether the present exposure status of op is acceptable or not at this point because there is no reference dose data of op . when we compared the daily exposure levels in the present neonate to those in adults , for example , the neonates may be 10-fold highly exposed to bpa than adults [ 1.2 ug / kg ( table 3 ) ] versus 0.13 ug / kg , which was estimated from korean adult urines . concerning edcs ' disposition , many researchers have analyzed edcs at adiposities . as lipophilic characteristics of edcs can induce chronic diseases even with buffering of acute toxicity , this issue has been emphasized . a recent spanish study showed that the most obese woman had the highest levels of np and pcbs in adipose tissue . however , it is not clear , yet , whether body fat contents affect phenol accumulation in the body . in the present study , we found positive associations between levels of mothers bmi before delivery and np or op levels . however , bpa levels showed opposite trend , that is , negative association with mothers bmis . for the reason of the opposite trend , we consider physiochemical difference in octanol - water partition coefficient value ( log p or kow ) between np and bpa ( 4.48 and 3.32 , resp . ) . in addition , people are simultaneously exposed to multiple edcs ; thus , we screened combined effects of bpa , np , and op on the present mothers ' and neonates ' health . however , the simple sum of total bpa , np , and op levels did not show any health risk . in order to study future and real combination effects of phenols or edcs , their weight for risks or reliable simulation of multiple exposures should be considered . referring pharmacokinetics of phenols in human or rhesus monkeys [ 28 , 29 ] , which may be mainly metabolized into urine as conjugated forms with glucuronyl or sulfonyl groups , we have used conjugated bpa for biomonitoring of bpa in urine or blood samples [ 14 , 15 , 30 ] . detoxification enzymes which metabolize free phenols into conjugated phenols are known to decrease in pregnant women . as most conjugated bpa was analyzed in urine , we analyzed bpa in some urines among the present mothers who donated urine ( n = 21 ) to confirm conjugation capacity in the present mothers . their conjugated bpa levels in urine were approximately half of those in colostrum ( mean std , 0.98 1.96 ug / l versus 2.44 3.68 ug / l , mean difference , 1.46 ug / l . ) when we compared the conjugated bpa levels in the present lactating mothers ' urine samples to those in other adults ' , they were quite lower than the others ( median , < 0.6 ng / ml versus 7.86 ng / ml ) . it may support pregnant women ' loss of detoxification enzyme activity , even though we consider the decline of bpa exposure due to years . we also studied exposure of routes of the phenols in mothers and found association between bpa levels and consumption of dairy products ( table 3 ) . the real parts , which people intake , directly contact to containers or packing materials rather than other food . like pcbs accumulation in perennial fish , accumulation of bpa in cow can be thought via food chain . interestingly , we also found a positive association between bpa levels and use of detergents for food . a recent report concerning the effect of detergents in the release of bisphenol a from polycarbonate baby bottles can support our result . referring reports of ap - related food [ 16 , 17 ] , we studied more other sources for aps , but we could not find any similar source , for example , fish , fruit , or fish oil . considering health risks of phenols , we found negative association between embryonic period in neonates and their colostrum np levels and suspect np induces embryonic instability . np showed some embryotoxicity in crustaceans and oysters , for example , low survival rates and poor embryonic and larval development [ 34 , 35 ] . however , experimental studies in mammals to clarify effects of np on embryonic period have been thoroughly performed , yet . thus , future enlarged epidemiological studies or experimental studies are required to confirm risks of np on embryonic stability . secondly , we found that levels of total np in sick mothers , particularly , toxemia - patients , were higher than those in health subjects ( table 4 ) . in addition , we found that the infant with congenital malformation of uvulas showed quite high levels of total aps in compared to others ' levels . thus , our finding should be further confirmed in enlarged studies , even though the present subjects well represent the korean women in childbed from similar proportion of toxemia or gestational complications . in conclusion , we found that most of neonates are exposed to bpa rather than np or op via colostrums . although current exposure levels of the phenols are safe based upon their tdis , we suggest continuous biomonitoring of them to clarify their unclear health risk on neonates and pregnant or gestation mothers .

facial trauma may be considered one of the most significant aggressions found in trauma centres due to the emotional consequences , the possibility of deformation , and the economical impact it causes in the health system . because of little protection and considerable exposure of the facial region , a great number of severe facial lesions often occur . maxillofacial fractures are more predominant in big cities due to intense traffic and high violence rate . different studies have shown the relationship between maxillofacial fractures , gender , age , and level of urban development [ 2 - 5 ] . as man evolved and developed more machinery to ease day to day living , the incidence and severity of trauma injuries to the face also increased . when trying to outline the epidemiological profile of facial traumas all over the world , many studies were carried out correlating social , urban , and rural changes as modifying agents of interpersonal relations , generating acts of physical violence both personal and collective , which are represented by interpersonal violence , traffic violence , domestic violence , and violence against women . men are more exposed to the trauma due to the greater number of automobile drivers , the practice of physical contact sports , in addition to a more intense social life inferring in higher consumption of alcohol and other drugs . in turn , some affirm that , especially over the last three decades , there has been a growing increase of traumas in women usually around 40 years old due to their increasing participation in activities that were previously male dominant . many studies show that , for children and the elderly , facial fractures are associated with either falls at home , or children 's games and activities [ 5,8 - 10 ] . considering the clinical aspects of the trauma , the high rate of complex nasal fractures and zygomatic orbital fractures are obviously related to the prominent position of these anatomical structures inside the facial skeleton , and their greater exposure to the external trauma . added to the facial trauma , it is possible to identify associated injuries and classify six organ systems related to these lesions : 1 ) brain , 2 ) chest , 3 ) abdomen , 4 ) pelvis , 5 ) spine , 6 ) limbs . there are considerable statistical differences in the occurrence of injuries associated among gender , age group , trauma mechanism , and type of fracture . the mechanism of these traumas may be through automobile accidents , falls , interpersonal violence , sports , and victims hit with objects . hands and arms are usually used by patients victims of trauma as protection against a facial lesion , while legs and thorax are usually directly impacted in car accidents or falls . the purpose of the present study was to identify the occurrence , types , and severity of associated injuries outside the facial region in patients diagnosed with facial fractures at hospital so vicente de paulo , passo fundo , brazil . this is a cross - sectional , retrospective observational study , which criteria was the inclusion of all medical records of patients with history of facial fracture assisted in the department of oral and maxillofacial surgery of hospital so vicente de paulo - passo fundo , brazil , along with the department of medical file and statistical service . the period established was ten years , starting on january 1 , 2001 and ending on december 31 , 2010 . data evaluated considered the etiological agent of the lesion , precedence , age , and gender of the patient as well as the site of fractures . patients with lesions exclusive to soft tissues of the face were not included in the study . the traumatic injuries associated to facial fractures were classified according to the different types . to analyze data , the medical records were divided according to the patients ' age group , in dozens . regarding precedence , the referential point was the city of passo fundo , and the classification " other locations " was assigned to patients from other cities due to the influence passo fundo has regionally , especially concerning health centres . the etiological agents were divided in seven groups : interpersonal violence , fall , automobile accidents , sports accidents , work accidents , others and when it was not described , this was classified as not informed . lesions by firearm , domestic violence , assaults , and physical fighting are included in the item " interpersonal violence " . included in the item " automobile accidents " are : being run over by motorcycles , bicycles , and automobiles . the group entitled " others " covers accidents with animals and removal of impacted teeth . the distribution of fractures site was divided in eight groups : zygoma , mandible , orbit , nose , maxilla , le fort ( i , ii , and iii ) naso - orbital - ethmoid and dentoalveolar fractures . the three types of le fort fracture were joined in one single item because of the few cases diagnosed . according to thorn et al . , the injuries associated to facial trauma were classified and divided by : upper limb , lower limb , skull , cranioencephalic trauma ( cet ) , thorax , spine , skin excoriation , and abdomen . cases with no injuries were included in the group " absent " . the project was submitted and approved by the research ethics committee of the hospital so vicente de paulo - passo fundo / rs , and by the committee of the university of passo fundo ( upf ) . the tabulated data were statistically analyzed using a chi - square test with significance level of 5% followed by the pearson 's correlation test in the program spss version 20 . the analysis of patients , who suffered maxillofacial trauma in this study from 2001 to 2010 , resulted in 1385 clinical medical records of different patients , recording a higher frequency of facial fractures in the age group between 20 and 39 years old . patients aged between 10 and 19 years old , and 40 and 49 years old also presented high rates of maxillofacial fracture occurrence , and this rate was higher in the male population ( 82.6% ) for all age groups ( table 1 ) . description of statistics of the studied population n = number of patients ; noe = naso - orbital - ethmoid . passo fundo - rs , considered reference for trauma in the northeast region of the state of rio grande do sul , brazil , presented lower frequency ( 35.2% ) of facial traumas , which were more common in patients from nearby cities ( 64.8% ) . the most common etiological agent among the analyzed cases was automobile accident ( 25.8% ) , followed by interpersonal violence and fall . the classification " not informed " obtained considerable numbers ( 22.3% ) due to failure to fill this item in the medical records ( table 1 ) . the most common affected site of facial fractures verified was mandibular fractures ( 34.7% ) , followed by zygomatic bone fractures ( 24% ) , and nose ( 22.8% ) . injuries associated to facial trauma presented skin excoriation ( 15.7% ) as main injury , followed by cet ( 12.8% ) . within the researched items , it is demonstrated that human body injuries associated to the patient victim of facial trauma occurred in about 35% of the cases ( table 1 ) . the correlation statistical analysis among the age group of patients who suffered associated body injuries revealed to be more relevant in the age group from 20 to 39 years old , adding 260 cases , followed by 10 to 19 years old ( 64 cases ) , and 40 to 49 years old ( 74 cases ) . the most named injury was skin excoriation followed by cet ( table 2 ) . distribution of injuries location depending on the patients ' ages n = number of patients ; ul = upper limb ; ll = lower limb ; cet = cranioencephalic trauma . it is possible to verify that among all medical records analyzed ( 1,385 ) , 506 cases of body injuries associated to facial trauma were processed . yet , in all of these cases , the most evident etiological agent was " automobile accident " representing 201 of the cases ( 39.7% ) , followed by " fall " ( 20.9% ) , and " interpersonal violence " ( 17.9% ) . the most evident injuries in relation to all types of etiological agents are skin excoriation representing 217 of the cases ( 15.7% ) , followed by cet ( 12.8% ) ( table 3 ) . distribution of injuries location depending on the etiological agent n = number of patients ; ul = upper limb ; ll = lower limb ; cet = cranioencephalic trauma . from the 1385 patients originally studied , the affected site that mostly showed facial fractures was the mandible representing 481 of the cases , followed by zygomatic trauma , and then the nasal . relating this data with injuries associated to facial trauma it is possible to conclude that skin excoriation was the most present injury in this study in practically all types of facial fracture . it is observed that in facial traumas in the midface of naso - orbital - ethmoid , le fort , and orbit types , the most associated injury was cet ( table 4 ) . distribution of injuries location depending on the affected sites n = number of patients ; noe = naso - orbital - ethmoid ; ul = upper limb ; ll = lower limb ; cet = cranioencephalic trauma . when analyzing the patients ' gender most affected by injuries associated to facial trauma , men prevailed representing 424 ( 37% ) injuries from 1144 cases . injuries that most affected this population were skin excoriation ( 16% ) , followed by cet ( 13% ) ( table 5 ) . distribution of injuries location depending on the gender n = number of patients ; ul = upper limb ; ll = lower limb ; cet = cranioencephalic trauma . with the analysis of 1385 medical records of patients with facial trauma , assisted at the hospital so vicente de paulo in the city of passo fundo - rs , brazil , it is possible to observe that most of them are male ( 82.6% ) . a study that analyzed 9543 patients with facial trauma allowed verifying a frequency of the male gender in these accidents with a proportion , mentioned by authors , of 2:1 male patients injured ( n = 6474 ) . other studies also confirmed the prevalence of the male gender in facial trauma . patients from this study in the age group from 20 to 40 years old presented a higher frequency of facial trauma ( 260 cases ) and associated body injuries . age groups between 20 and 49 years old are the most common relating to facial trauma ; automobile accident and interpersonal violence are the most frequent agents causing these fractures , still regarding the present study . the average age of patients with associated body injuries of the studied population was of 29.8 years old , once again in favour of the results of the present study . the most prevalent etiological agent that caused body injury associated to facial trauma was automobile accident ( 39.7% ) . in this study , the second cause was interpersonal violence that represented 17.9% of the cases of body injuries associated to facial trauma . a study that analyzed 325 patients showed that car accidents are responsible for about 2/5 of body injuries associated to facial trauma , followed by interpersonal violence ( 17% ) . interpersonal violence , which can be framed in the matter of urban violence , is associated to emotional and socioeconomic conflicts , especially among younger population . relating to facial trauma , reducing interpersonal violence will be more complicated than , for example , decrease these occurrences for automobile accidents , because the tendency of urban violence and social conflicts is to increase . the lack of investments from the government in education and public safety concerns and worsens this situation even more . an automobile accident causes a more random pattern of mechanical trauma , with forces distributed to the entire body , which is conducive to injury to multiple parts of the body . based on the results of the present study , concomitant injuries in areas other than the face in patients with facial fractures should be expected first and foremost in connection with high - speed trauma mechanisms and in association with severe facial injuries . trauma that involves sufficient energy to fracture the bones of the facial skeleton is also likely to distribute a substantial amount of force to other parts of the body , and thus cause injury . . the reasons for this high frequency are difficult to postulate but may be due to the factors mentioned below . inadequate road safety awareness , unsuitable road conditions without expansion of the motorway network , violation of speed limits , old vehicles without safety features such as anti - burst locks and energy absorbing materials , failure to wear seat belts or helmets , entry in to opposite traffic lane , violation of the right of the way , violation of the highway code , use of alcohol or other intoxicating agents , behavioural disorders and socio - economical insufficiencies of some drivers . the most prevalent affected site was the mandible representing 481 of the cases , followed by zygomatic and nasal . these sites are featured mainly because they are prominent within the facial skeleton and more exposed to the external trauma . studies showed high rates of car accidents that tend to present mandible fracture as most frequent fracture site . the z ygoma prevailed in 332 of the cases , standing as second most affected site . when compared to the previous study , in the same location between 1999 and 2000 , these data revealed some disagreements , both in the frequency of most affected face site in the occasion , the z ygomatic - and in the difference between automobile accident and interpersonal violence - greater in the previous study . this differences are mainly due to the different time period analyzed , and above all it is observed a decrease in the rate of facial fractures occurred by automobile accident and an increase of interpersonal violence as etiological factor . these data may suggest a greater awareness by the local population regarding traffic risks . preventive measures , such as the obligatory wearing of a crash helmet and seat belts , better enforcement of the law regarding " drinking and driving " , educating people about the dangers of all - terrain injuries and providing proper safety guidelines before the purchase of a vehicle have been shown to significantly reduce the number of road traffic accidents . the nasal region was the most compromised in studies that evaluated victims of car accidents , and analyzed victims of interpersonal violence . thus , it is possible to affirm that different traumatic etiological agents may lead to lesions with similar aspects and distributions . the injury associated to facial trauma mostly found in this study were skin excoriation , totalizing 267 of 616 injuries from the total of 1688 fractures in the group of 1385 patients , followed by cet , which totalized 220 cases . on the other hand , it was reported in literature that 55% of 200 patients with facial fracture had cet . in a retrospective study that analyzed 42 patients with naso - orbital - ethmoid fractures , in the period from january 1987 to march 2002 , it is possible to assess that the risk group for these fractures is composed by male individuals , from 21 to 40 years old , particularly involved in automobile accidents , and that the main associated injury was the cet . facial lesions in 580 patients who presented 935 mandibular fractures determined that 46.6% of these fractures were associated to lesions in soft tissues , with prevalence of head injuries , facial laceration ( 29.8% ) , and ocular lesions ( 5.7% ) . the epidemiological investigation of facial traumas along with associated body injuries enables the outlining of risk circumstances , as well as the characteristics of individuals who are more susceptible to facial trauma in the city of passo fundo and nearby region . this study also allowed assessing the most affected facial fractures sites , as well as identifying the body injuries associated to this trauma . the maxillofacial surgeon who performs the emergency service must be aware of the fact that about 25% of patients with facial fractures have lesions in other parts of the body , after all , this is the professional who is in the front line performing the primary evaluation of patients who suffered fractures in the facial bones . in this study , the prevalence of concomitant injuries in this series serves as a reminder of the acuity of these patients and the importance of a multidisciplinary approach to the trauma patient . according to the results of this work , it was possible to verify and conclude that in 35% of the cases of facial fracture there was an associated body injury . the main etiological factors for this association were automobile accidents , falls , and interpersonal violence . most fractures were mandibular , and the main associated body injury observed were skin excoriation , however , when considered the fractures of the middle third the main associated body injury were traumatic brain injuries . concomitant injuries in areas other than the face should be expected first and foremost after high - speed trauma mechanisms and in association with severe facial fractures . the results underscore the importance of multiprofessional collaboration in diagnosis and sequencing of treatment who have sustained facial fractures . the authors would like to thank iris penz ( hospital so vicente de paulo , passo fundo , brazil ) for your help in editing this manuscript .

emphysematous pyelonephritis ( epn ) is a necrotizing infection characterized by the presence of gas within the kidney and perinephric tissue . the condition , usually occurring in diabetic patients , is rare and often life threatening . the enteric gram - negative bacilli such as escherichia coli , enterobacter , klebsiella , and proteus account for most of the reported cases , with e. coli accounting for 60% . gas formation in epn is due to pathogenic bacteria causing mixed acid fermentation in a hyperglycemic environment in tissues that are ischemic . this results in tissue destruction and encourages purulent infection and inhibition of removal of locally produced gas . with advancements in imaging techniques and availability of newer antibiotics , medical treatment with or without percutaneous drainage is becoming an acceptable alternative for radical surgery.[79 ] several cases have been reported where diabetic patients with epn were managed successfully with medical treatment.[1012 ] here , we report a newly detected diabetes patient with epn who was successfully managed by non - surgical treatment . a 70-year - old woman presented with 4-day history of fever , right flank pain , polyurea , and altered sensorium . she had been detected to have diabetes mellitus about a month back and was taking oral antidiabetic medication . the patient was ill looking , agitated , and talking irrelevant ; she was febrile ( temperature 100f ) and tachypnic ( respiratory rate 34 per minute ) ; her heart rate was 110 bpm and blood pressure was 90/60 mmhg . on systemic examination , air entry was reduced and crepitations were audible more on the right side ; suprapubic and right renal angle tenderness was present and no abdominal mass was palpable . initial laboratory evaluation included complete blood count ( hemoglobin 9.9 g% , total leukocyte count 9500/l with 86% neutrophils , and platelet count of 36,000/l ) , kidney function tests ( blood urea 95 mg / dl and serum creatinine 4.6 mg / dl ) , random blood glucose ( 408 mg / dl ) and arterial blood gas analysis ( ph 7.37 , sao2 89.5% , pco2 18.3 mmhg , po2 56.4 mmhg , and hco3 10.4 usg abdomen revealed left kidney size of 11.1 4.4 cm with mild increased echo pattern . ncct of brain was normal ; cerebrospinal fluid examination , performed in view of altered sensorium , was normal . repeated urine and blood culture samples taken during the hospital stay failed to grow any organism . computed tomography ( ct ) of the abdomen revealed the presence of gas in the right pelvicalyceal system and renal parenchyma , with air and debris in dilated right ureter . the diagnosis of epn was made and the patient was managed with oxygen inhalation , intravenous fluids in the form of normal saline , and insulin infusion to achieve euglycemia . empiric intravenous antibiotics in the form of pipericillin , tazobactum , and metronidazole were administered . because of persistent fever , hypotension and worsening renal functions , the antibiotic spectrum was broadened in the form of addition of ciprofloxacin and vancomycin . the patient 's clinical condition started improving around the 4 day of treatment and ncct on the 5 day revealed marked improvement in the form of decrease in the size and extent of epn [ figure 1b ] . after 3 weeks , another ncct abdomen revealed complete disappearance of features of epn [ figure 1c ] . after this , parenteral antibiotics were stopped and the patient was put on oral third - generation cephalosporin ( cefpodoxime proxitel + clavulanate ) and ciprofloxacin . she was on oral diet and was discharged on two doses of premixed ( 30/70 ) insulin at the end of 4 weeks . when reviewed after two more weeks ( a ) non - contrast ct ( ncct ) of abdomen at admission revealing gas in the right pelvicalyceal system and renal parenchyma , with air and debris in dilated right ureter suggestive of epn ; ( b ) ncct on the 5th day revealing decrease in the size and extent of epn ; ( c ) ncct after 3 weeks revealing complete disappearance of features of epn lab parameters of the patient during hospital stay first described in 1898 , epn is an acute necrotizing parenchymal and peri - renal infection caused by gas forming uropathogens . four factors have been proposed to have a role in the development of epn : gas forming bacteria , high blood glucose , damaged tissue perfusion , and impaired immune response . the disease predominantly affects women , with the left kidney being more frequently involved whereas both kidneys are involved in about 510% of the reported series.[51315 ] though 90% of the reported cases have occurred in diabetic patients , epn has also been reported in debilitated , alcoholic , and immunocompromised patients . most patients with epn are severely ill with fever , chills , flank pain , lethargy , confusion associated with medical problems . a typical feature of epn seen on conventional radiography is the presence of gas outlining ureters and pelvicalyceal system . ultrasound findings include high - amplitude shadowing along the non - dependent surfaces , causing obscuration of the posterior structures . the gas appeared as a hypoechogenic area surrounded by an echogenic ( calcific ) rim and more echogenic central stone , thus giving the appearance of a target sign . ct scan is the investigation of choice for not only making a proper diagnosis but also planning the treatment option . , based on ct appearance , divides epn into two types with different prognostic significance . type 1 is characterized by parenchymal destruction with streaky or mottled gas collection but no fluid collection . reduced immune response limits the formation of pus collection and this leads to the spread of the inflammation culminating in a fulminant course of the disease ; it carries a high mortality of around 70% . type 2 is characterized by bubbly or loculated gas within the parenchyma or collecting system with associated renal or peri - renal fluid collection . a better immune response results in the formation of pus in the kidney , leading to a slower course of the disease and better prognosis with a mortality of around 16% . a more detailed staging has been put forward by haung et al . as follows : class 1 gas in the collecting system ; class 2 gas in parenchyma without extension into the extrarenal space ; class 3a extension of gas to perinephric space ; class 3b extension of gas to paranephric space ; and class 4 bilateral epn or single kidney epn . our case is considered to be of group 2 according to either classification because the gas in the renal pelvis was confined to the kidney itself . patients initially presenting with thrombocytopenia , acute renal function impairment , disturbance of consciousness , and shock have very high mortality . our patient had all these adverse features at presentation , but responded well to conservative treatment . traditionally , the consensus is that mere medical treatment may not be effective and prompt nephrectomy is necessary because the mortality rate in patients treated with antibiotics alone is around 40% . success of treatment is 66% in those treated with percutaneous nephrostomy and 90% in those treated with nephrectomy . with the availability of ct and better antibiotics , a combination of better antibiotics and image - guided drainage has been put forward as an initial mode of treatment . asgari reported the case of a 45-year - old diabetic woman on oral hypoglycemic agents who had presented with epn . similarly , many individual cases have been described where a conservative treatment has been found successful with a combination of good metabolic control and antibiotics . we managed the present case conservatively with aggressive medical treatment including good and rapid control of glucose , hypoxia , sepsis , and renal failure , and the patient had a favorable outcome . we thus believe that nephrectomy is not the preferred treatment anymore for all cases of epn .

drug - induced hepatotoxicity is a major contributor to the high attrition rates of drug candidates during preclinical and clinical drug development . it is also responsible for many postlaunch withdrawals and labeling restrictions for drugs which have successfully gone through the discovery and development process . assessment of hepatotoxicity remains difficult because of challenges associated with in vivo models and the high cost and limited availability of liver tissue for in vitro studies . current in vitro models for assessing hepatotoxicity are limited by ( a ) scarcity , variability , and short life span in culture of primary human hepatocytes ; ( b ) lack of metabolic activity in widely used liver cell lines such as hepg2 ; and ( c ) the complex long - term protocols required to differentiate progenitor cells . in recent years , heparg cells have emerged and are being increasingly adopted as an alternative to hepg2 cells and primary hepatocytes for in vitro hepatotoxicity studies , overcoming many of the limitations associated with existing hepatocyte cellular models . the heparg human cell line was established from a tumor of a female patient suffering from chronic hepatitis c infection and hepatocarcinoma . when passaged at low density , they are able to recover and differentiate into both hepatocytes and biliary epithelial cells and are thus considered to be progenitor cells . gene expression profiling has shown that heparg cells are remarkably close to human hepatocyte populations . unlike other immortal hepatic cell lines such as hepg2 , heparg display many characteristics of primary human hepatocytes , including cytochrome p450 mediated metabolism , transporter functions , and expression of key nuclear receptors known to play important role in liver function following drug exposure . accordingly , these cells have served as an effective surrogate for primary human hepatocytes in a wide variety of liver - specific functional assays [ 7 , 1113 ] . initially , heparg cells required several weeks of culture to bring them to a differentiated state ; however , heparg cells have recently become available in a ready - to - use cryopreserved differentiated format which has shown promise for drug metabolism studies . high content analysis ( hca ) , an imaging - based quantitative cellular analysis technology , enables multiparametric detection of events in individual cells in situ and is well - suited for high - throughput assessment of hepatotoxicity . pioneering work has extensively validated this technique for analysis of hepg2 cells and primary hepatocytes [ 1619 ] . this study aimed to characterize the cryopreserved differentiated heparg cells for use as human hepatocyte surrogates in high content analysis applications and to determine if imaging - based detection of cyp3a4 activity is feasible . specific goals were ( a ) to determine if cryopreserved differentiated heparg cells retain key functional hepatocyte characteristics , ( b ) to determine if these cells are amenable to multiparametric hca under conditions where cyp3a4 activity is retained , and ( c ) to determine optimal assay conditions for the application of these cells to imaging - based cyp3a4 expression studies and multiparametric hepatotoxicity assessment . cryopreserved heparg cells ( catalog # mmhpr116 ) , heparg thawing / plating medium supplement ( catalog # mmadd671 ) , heparg induction medium supplement ( catalog # mmadd641 ) , and heparg culture medium supplement ( catalog # mmadd621 ) were from emd millipore ( billerica , ma ) . williams e medium ( wem ) and glutamax were purchased from in vitro technologies , inc . hepg2 cells ( catalog # hb-8065 ) were obtained from atcc ( manassas , va ) . for hepg2 culture , mem / ebss ( catalog # sh3024401 ) , fetal bovine serum ( catalog # sh3007103 ) , nonessential amino acids ( catalog # sh3023801 ) , sodium pyruvate ( catalog # sh3023901 ) , and penicillin / streptomycin / glutamine ( catalog # sv3008201 ) were obtained from thermo fisher scientific , inc . 96-well polystyrene tissue culture plates and rat tail collagen type i were obtained from bd biosciences ( san jose , ca ) . dimethyl sulfoxide ( dmso ) , hoechst 33342 nuclear stain , formaldehyde , triton x-100 , periodic acid schiff ( pas ) reagents , rifampicin ( rif ) , and omeprazole ( ome ) were purchased from sigma - aldrich ( st . louis , mo ) . elisa kits for detection of albumin and alpha-1-antitrypsin were purchased from bethyl laboratories , inc . gsh - glo glutathione and p450-glo assay kits were purchased from promega corporation ( madison , wi ) . rabbit anti - human cytochrome p450 enzyme cyp3a4 polyclonal antibody ( catalog # ab1254 ) , mouse anti - cytokeratin 19 monoclonal antibody ( catalog # mab3238 ) , donkey anti - rabbit igg antibody , cy3 conjugate ( catalog # ap182c ) , and donkey anti - mouse igg antibody , fitc conjugate ( catalog # ap192f ) , were from emd millipore ( billerica , ma ) . mitotracker green fm ( catalog # m-7514 ) , monochlorobimane ( mbci ) ( catalog # m-1381mp ) , celltracker red cmtpx ( catalog # c34552 ) , tetramethylrhodamine , methyl ester , perchlorate ( tmrm ) ( catalog # t668 ) , and alexa fluor 568 phalloidin ( catalog # cryopreserved heparg cells were thawed and cultured in collagen coated 96-well plates according to the supplier 's protocol . heparg cells were allowed to attach for 6 hrs , at which time the heparg thawing / plating medium was renewed . hepg2 cells were thawed and cultured according to vendor 's instructions with media replenishment 1 day after thaw and every 3 days thereafter . following 48 h culture after thaw , heparg and hepg2 cells cultured in 96-well plates at a seeding density of 50,000 cells per well were assessed for a panel of functional activities associated with human hepatocytes , using well - established reagents and protocols . periodic acid - schiff staining was performed according to manufacturer 's instructions in order to identify the presence of glycogen within the cell cultures . secretion of albumin and alpha-1-antitrypsin by hepg2 and heparg cells was assessed using elisa kits for each , with tests performed according to manufacturer 's instructions , using albumin and alpha-1-antitrypsin standards provided with the test kits to enable quantitation of each in control samples of tissue culture media which had not been in contact with cells , or in tissue culture media aspirated from hepg2 or heparg cell cultures following 48 h culture after thaw . levels of glutathione ( gsh ) in hepg2 and heparg and its depletion in response to treatment with buthionine sulfoximine ( bso ) were assessed using a luminescence - based assay performed according to manufacturer 's instructions for adherent cell cultures , whereby tissue culture medium is removed from the cells prior to incubation of the cells with luminescent detection reagents . basal levels of cytochrome p450 ( cyp3a4 ) enzyme activity were measured after culturing the cryopreserved heparg or hepg2 cells for 3 days . for induction studies , heparg and hepg2 cells were exposed to the prototypical cyp3a4 inducer rifampicin ( rif ; 10 um ) for 72 hrs , starting the induction on day 3 after plating the cells . basal and induced cyp3a4 activities were measured using p450-glo assays , according to the manufacturer 's instructions . induced cyp3a4 enzyme activity was calculated as fold increase over dmso controls . for multiplexed hoechst / ck-19/cyp3a4 staining , at the end of the induction period , plates were removed from the incubator and the cells were immediately fixed with 100 l of prewarmed fixation solution containing 7.4% formaldehyde for 30 min at room temperature . following fixation , each well was gently washed twice with a cell permeabilization buffer containing 0.25% triton x-100 . a primary antibody working solution containing rabbit anti - cyp3a4 ( 1 : 1000 dilution ) and mouse anti - cytokeratin-19 ( 1 : 500 dilution ) antibodies was prepared in this buffer and 50 l was applied to each well ; then plates were incubated for 1 h at room temperature . a secondary antibody working solution containing cy3-donkey anti - rabbit antibody ( 5 g / ml ) , fitc - donkey anti - mouse antibody ( 5 g / ml ) , and hoechst 33342 nuclear stain ( 10 g / ml ) was prepared and 50 l was applied to each well for 1 h at room temperature . cells were then washed twice with pbs , and plates were then stored , sealed , and protected from light at 4c until image acquisition . for other stainings , mitotracker green fm was added to live , unfixed heparg cells at a concentration of 0.1 m and incubated for 30 minutes prior to imaging ; mbci dye was added to live , unfixed heparg cells at a concentration of 80 m and incubated for 10 minutes prior to imaging ; celltracker red cmtpx was added to live , unfixed heparg cells at a concentration of 0.5 m and incubated for 30 minutes prior to imaging ; tmrm dye was added to live , unfixed heparg cells at a concentration of 0.1 m and incubated for 30 minutes prior to imaging ; alexa fluor 568 phalloidin was added to fixed heparg cells at a concentration of 0.165 m and incubated for 60 minutes prior to imaging . assay plates were imaged with a ge in cell analyzer 1000 high content imaging system and images were analyzed with ge in cell analyzer 1000 workstation ( 3.7 ) software , utilizing the multitarget analysis algorithm to segment cellular features based on size and fluorescence intensity - related criteria . imaging was performed using a 20x objective lens , acquiring 10 fields of view for each wavelength per well . image analysis parameters were optimized in ge workstation ( 3.7 ) software for accurate and robust cell and feature segmentation , using representative images of treated and untreated cells . automated image analysis was performed on each well , collecting data on a cell - by - cell basis , which was then averaged within the application , and the reported well - by - well summary was used for subsequent analysis . for all assays , biological replicates from three separate wells were averaged to obtain the mean and standard error of the mean for each treatment dose . to determine drug - induced cellular changes , student 's t - test ( two - tailed distribution , two - sample equal variance , p < 0.05 ) was used to determine the significance of responses . heparg cells represent an attractive option for hepatotoxicity applications because they retain many features of primary human hepatocytes which are not present in other hepatic cell lines , including activity of the critically important drug metabolizing enzyme cyp3a4 . this study characterized the cryopreserved differentiated form of the cells and compared them to the widely used hepg2 cell line with regard to phenotypic features characteristic of human hepatocytes . while high content analysis represents a powerful technique for hepatotoxicity and drug screening applications [ 1619 , 21 , 22 ] , the quality of hca data is largely determined by the effectiveness of image analysis algorithms at accurately identifying and segmenting cellular features of interest . since heparg differentiate into a complex mix of hepatocyte and biliary epithelial cells and since established protocols for culture of heparg cells generally require relatively high cell seeding densities which may confound image segmentation algorithms , we hoped to establish an optimal set of conditions for heparg culture which would enable quantitative hca without compromising cyp3a4 activity in these cells . we first characterized cultured cryopreserved differentiated heparg with regard to a panel of functional markers associated with primary human hepatocytes , comparing heparg with hepg2 cells ( figure 1 ) . using a periodic acid - schiff ( pas ) staining system with a hematoxylin nuclear counter stain , pas positive substances stain pink and nuclei are blue . the normal liver contains a large amount of glycogen , leading to hepatocytes staining intensely pink with a pas stain . figure 1(a ) shows that heparg cells stain strongly pink with pas staining , indicative of high glycogen content , whereas hepg2 cells stain much less strongly for glycogen , with the blue nuclear counter stain dominating ( figure 1(a ) ) . albumin secretion is characteristic of hepatocytes , and both heparg and hepg2 cells were found to secrete significant amounts of albumin into tissue culture medium ( figure 1(b ) ) , although at equivalent cell seeding densities heparg were observed to secrete significantly more albumin than hepg2 cells ( 1.8-fold greater than hepg2 ; p > 0.05 ; figure 1(b ) ) . similar to albumin , both heparg and hepg2 cells released significant amounts of alpha-1-antitrypsin into tissue culture medium ( figure 1(c ) ) , with heparg releasing significantly more than hepg2 cells seeded at an equal density ( 2.0-fold greater than hepg2 ; p > 0.05 ; figure 1(c ) ) . hepatocytes also synthesize glutathione , an important cellular feature involved in regulating cellular metabolism and responses to several types of toxic challenge . both heparg and hepg2 cells synthesize glutathione ( figure 1(d ) ) although untreated heparg cells were found to synthesize significantly more glutathione than the equivalent number of hepg2 cells ( p > 0.05 ; figure 1(d ) ) . in both heparg and hepg2 cells , glutathione synthesis was significantly depleted ( p > 0.05 ; figure 1(d ) ) by treatment with buthionine sulfoxide ( bso ) . taken together , these data suggested that cryopreserved differentiated heparg cells , more so than hepg2 , exhibit multiple phenotypic characteristics associated with primary hepatocytes . however , for drug safety and hepatotoxicity applications , of greater importance is cellular metabolic function , particularly activity of cyp3a4 and other enzymes associated with drug metabolism . as the primary drug metabolizing enzyme isoform in humans , expression and activity of cyp3a4 in cellular hepatocyte models are highly desirable . we first determined the effect of cell seeding densities on basal cyp3a4 enzyme activity in heparg , comparing these cells with hepg2 cells ( figure 2(a ) ) . for this , cells were plated at three different seeding densities : 25,000 , 50,000 , and 75,000 cells per well in 96-well plates . the basal cyp3a4 activity was determined on day 3 after thawing and plating the cells . in accordance with previous observations , basal activity of cyp3a4 in hepg2 cells was extremely low , reflecting a well - known limitation of this cell type . at all three seeding densities tested , heparg cells exhibited significantly greater basal cyp3a4 activity than hepg2 cells ( figure 2(a ) ) . in heparg cells , basal cyp3a4 activity appeared to be closely linked to cell seeding density , with basal cyp3a4 activities in these cells increasing significantly as the seeding density was increased ( figure 2(a ) ) . highest basal cyp3a4 activity was observed at a seeding density of 75,000 cells per well , representing 144% of the basal activity observed at 50,000 cells per well and 215% of the basal activity observed at 25,000 cells per well ( figure 2(a ) ) . to establish the potential of each cell type for cyp3a4 induction , we treated cells with the prototypical cyp3a4 inducer rifampicin ( 10 m ) for 72 hours and compared effects of this drug to dmso - treated vehicle control samples ( figure 2(b ) ) . in hepg2 cells , no statistically significant effects of rifampicin treatment were observed at any seeding density ( figure 2(b ) ) . in contrast , cyp3a4 was potently induced by rifampicin in heparg cells at each seeding density . compared to dmso controls , rifampicin induced cyp3a4 activity 22-fold ( p < 0.05 ) at cell seeding density of 25,000 per well and 33-fold and 31-fold ( both p < 0.05 ) at seeding densities of 50,000 and 75,000 cells per well , respectively ( figure 2(b ) ) . these data indicated firstly that the cryopreserved , differentiated form of heparg cells retains the high levels of expression and inducibility of cyp3a4 of the parental cell line and secondly that cyp3a4 activity and inducibility in these cells are linked to cell seeding density , with the higher seeding densities of 50,000 and 75,000 cells per well having parity with regard to cyp3a4 fold induction . to further characterize the cryopreserved differentiated form of heparg cells as a model system and in particular to determine the suitability of these cells for long - term in vitro applications , we measured the basal cyp3a4 activity over a time period of 12 days after plating the cells for seeding densities of 50,000 and 75,000 cells per well ( figure 3 ) . the cells showed high basal cyp3a4 activity in suspension immediately after thawing the cells on day 0 ; however , at both seeding densities , cyp3a4 activity then decreased by approximately 50% during days 1 and 2 in culture before recovering to day 0 levels from day 3 onwards ( figure 3 ) . basal cyp3a4 activity at each seeding density increased steadily over time from day 3 . by day 12 , basal cyp3a4 at both seeding densities stabilized at approximately 200% of day 0 levels ( figure 3 ) . these data indicate that the cryopreserved differentiated form of heparg is suitable for long - term in vitro studies and suggest that users ought to take account of the early declines in basal cyp3a4 activity during the first two days of plating into consideration when designing experiments . in all experiments in this study , we chose to wait until day 3 before treating the cells with test chemicals . having established that heparg cells express active cyp3a4 , we then sought to examine activity of two other cyp isoforms known to be highly expressed in hepatocytes and involved in human drug metabolism , namely , cyp1a2 and cyp2c9 . in hepg2 cells , no basal or induced activity of cyp1a2 or cyp2c9 was detectable . in heparg cells , under basal conditions , cyp1a2 activity was found to be weak but was found to be potently induced by treatment with 50 m omeprazole at each seeding density tested ( figure 4(a ) ) . although total induced activity of cyp1a2 was greatest at a seeding density of 75,000 cells per well , the greatest fold change over dmso control was observed at a cell seeding density of 50,000 cells per well ( 53-fold increase ; p < 0.05 ; figure 4(a ) ) . in contrast to cyp1a2 , basal cyp2c9 activity was readily detectible in heparg cells , but no induction was observed using the prototypical inducer rifampicin ( figure 4(b ) ) . together , these data indicate that although the drug metabolizing capability of cryopreserved differentiated heparg cells is not fully equivalent to primary human hepatocytes , these cells express strongly inducible cyp3a4 and cyp1a2 , and this cyp activity of heparg clearly distinguishes them from hepg2 cells . this feature of cryopreserved differentiated heparg is likely to be of great benefit for studies directly examining agents causing hepatotoxicity . for example , the activation of mycotoxin aflatoxin b1 is mediated in the liver by cyp3a4 and cyp1a2 , likely explaining the recent report that this agent is significantly more cytotoxic in cultures of heparg cells compared to hepg2 cells . clearly , the presence of active cyp isoforms in heparg cells confers significant advantages over nonmetabolically competent hepatocyte cellular models . we next sought to characterize the utility of cryopreserved differentiated heparg cells for imaging - based high content analysis applications . differentiated heparg cells stop proliferating and form colonies exhibiting a morphology resembling that of normal hepatocytes in primary culture with a dense cytoplasm and carboxy dichlorofluorescein diacetate- ( cdfda- ) staining bile canaliculus - like structures surrounded by more flattened and clearer epithelial cells corresponding to biliary cells [ 27 , 28 ] . we developed a multiplexed cell staining protocol to characterize the cryopreserved differentiated heparg cells used in this study , combining hoechst 33342 dye to stain all cell nuclei within the cultures , an antibody against cytokeratin 19 ( ck-19 ) , a marker of epithelial cells , and an antibody against cyp3a4 . this staining approach enabled us to confirm the existence of both hepatocyte and biliary epithelial cells within cultures of cryopreserved differentiated heparg . figure 5 shows images of cells seeded at 50,000 cells per well , cultured for 3 days , then treated with dmso vehicle control or 10 m rifampicin , and stained using the staining cocktail described . by examining images acquired from vehicle - treated cells ( figures 5(a)5(d ) ) , it becomes apparent that two distinct cell populations exist within the cultures , indicated by the presence or absence of ck-19 expression . levels of ck-19 in cells expressing this marker are relatively homogeneous across the population ( figures 5(b ) and 5(d ) ) . in contrast , in vehicle - treated cells , a small population of cells stain strongly positive for cyp3a4 , whilst the cyp3a4 protein is only weakly present in the remaining ck-19-negative cells ( figures 5(c ) and 5(d ) ) . we hypothesized that this weak cyp3a4 staining in vehicle - treated cells represented a basal level of cyp3a4 expression which may be upregulated by treatment of heparg cells with a cyp3a4 inducer . this was confirmed by examining images of rifampicin - treated heparg cells , where it was clearly observed that a dramatic upregulation of cyp3a4 expression occurred in response to this drug ( figures 5(g ) and 5(h ) ) . having performed qualitative assessment of images indicating that cyp3a4 induction by rifampicin is detectable by immunocytochemical methods , we then sought to determine if hca image analysis software could be applied to quantify the visually observed effects of the drug . through application of the multitarget algorithm within ge in cell investigator software , we generated cell segmentation masks for hoechst 33342 and cyp3a4 stains in vehicle and rifampicin - treated heparg cells ( figure 6 ) . we found that , at the highest cell seeding density of 75,000 cells , the image analysis algorithm had difficulty segmenting individual cells due to their close proximity ; however , no such issues were observed at seeding densities of 50,000 or 25,000 . within figure 6 , which shows cells seeded at 50,000 cells per well , it can be observed that , when properly optimized for the features of interest , the image analysis algorithm very accurately detects hoechst stained nuclei ( blue outlines ) , cyp3a4-positive cells ( green outlines ) , and cyp3a4-negative cells ( red outlines ) ( figures 6(e)6(h ) ) . accurate image segmentation is required to enable accurate quantitation of the features of interest within images . in this instance , we were particularly interested to examine the numbers of cells expressing cyp3a4 , the intensity of the cyp3a4 staining , and features derived from the nuclear staining , such as cell count and nuclear size . we first quantified the percentage of cyp3a4 expressing cells relative to the total heparg cell population as assessed by automated counting of hoechst - stained nuclei ( figure 7(a ) ) . this analysis showed that hca assessment of the percentage of cyp3a4-expressing cells data correlates well with the cyp3a4 basal and induced activity data generated using a luminescence - based p450 assay ( figure 2 ) . in vehicle - treated cells , the percentage of cyp3a4-expressing cells as assessed by hca was closely linked to cell density : at 25,000 cells per well , 14% of cells were cyp3a4-positive , increasing to 34% at 50,000 cells per well and 40% at 75,000 cells per well ( figure 7(a ) ) . rifampicin treatment significantly ( p < 0.05 ) increased the percentage of cyp3a4-expressing cells at all seeding densities , to 30% at 25,000 cells per well , 60% at 50,000 cells per well , and 61% at 75,000 cells per well ( figure 7(a ) ) . this novel approach provides evidence that high content analysis technology can be effectively employed to detect cyp3a4 in single cells in situ . when reviewing the heparg staining images and cellular segmentation data , shown in figures 5 and 6 , it became apparent that the hepatocyte and biliary epithelial subpopulations within the heparg cultures have different - sized nuclei , with the hepatocyte population having smaller nuclei and the biliary epithelial cells having larger nuclei . since the hca analysis of hoechst staining captured information on nuclear size , we were able to determine that the hepatocyte and biliary populations within heparg may be separated from one another during analysis by filtering the data based upon nuclear area . we created an image analysis filter applied at a nuclear area of 125 m so that cells with nuclei on either side of this filter could be analyzed separately and applied this to the data in figure 7(a ) . cells with nuclei with area lesser than 125 mare hereafter referred to as small nuclei and those with nuclei greater than 125 m as we observed that , under each condition tested , the cyp3a4-expressing population of heparg cells is predominantly ( 8590% ) made up of small nuclei cells ( figure 7(b ) ) . this suggests that analyzing only small nuclei cells within heparg cultures provides a greatly enriched population of hepatocytes for analysis and excludes the biliary epithelial cells . a small number of large nuclei , cyp3a4-expressing cells were also responsive to rifampicin ( data not shown ) , indicating that these were also hepatocytes . slight adjustments to the nuclear size filter may have slightly improved the detection percentage of the hepatocyte population , but our primary interest here was to exclude nonhepatocytes from analysis of hepatocyte - specific endpoints . in addition to assessment of the numbers of cyp3a4 expressing cells , we also sought to determine if changes in cyp3a4 expression levels could be quantified via hca . figure 8 shows analysis of the cellular intensity of cyp3a4 staining in small nuclei heparg cells . these data show that , in addition to quantifying numbers of cyp3a4-expressing cells , hca analysis of cyp3a4 can be used to sensitively detect changes in expression levels as indicated by changes in the cellular fluorescence intensity . in vehicle - treated cells , the highest intensity of cyp3a4 expression was observed at a seeding density of 75,000 cells per well , representing 106% of the fluorescence intensity observed at 50,000 cells per well ( not statistically significant ) and 148% ( p < 0.05 ) of that observed in cells seeded at 25,000 cells per well ( figure 8) . rifampicin treatment significantly ( p < 0.05 ) increased the intensity of cyp3a4 expression at all seeding densities , evoking at 1.8-fold increase in cells seeded at 25,000 cells per well , a 2.2-fold increase at 50,000 cells per well , and a 2.1-fold increase at 75,000 cells per well ( figure 8) . thus , image analysis - based techniques can be used to effectively generate sensitive , quantitative , and cell - by - cell data on cyp3a4 from intact heparg cells in situ . based on these data , we have found 50,000 cells per well to be the optimal seeding density for hca assessment of heparg in 96-well plates . there are significant advantages to be derived from our observation that it is possible to algorithmically separate the heparg hepatocyte population from the biliary epithelial population during image analysis . firstly , as with other heterogeneous or cocultured cell populations , it is imperative to be able to study cell - specific functions in isolation , so as not to contaminate cell - specific data with data from other cells residing within the culture milieu . this is a limitation of assays which rely on whole well analysis or cell lysis steps during sample preparation . image analysis renders it possible to study individual cell types in situ without the need to disrupt the culture and/or physically sort cells . high content analysis of cellular images is known to be a powerful technique for isolating different cell populations within complex cultures ; however , this approach generally relies on inclusion of cell - type - specific markers in order to segment each population separately . as most automated microscopes and hca instruments have a limited number of fluorescence channels ( typically 3 - 4 ) available per assay , multiple channels are frequently used simply for cell - specific identification markers and the requisite nuclear stain and thus are not available to study other cellular events . indeed , when commencing this study , we expected that inclusion of hepatocyte - specific ( cyp3a4 or perhaps hnf4 ) and epithelial - specific ( ck-19 ) markers would be required to separate the subpopulations of hepatocyte and biliary epithelium cells within heparg cultures during analysis . however , the differences in nuclear size between these populations observed and reported here render these additional stains unnecessary , as nuclear size can be used to effectively separate these populations . this saves both time and resources and frees up fluorescent imaging channels to examine other features of interest within the cells . as proof of concept for other imaging - based assessments that could be performed in cryopreserved differentiated heparg cells and multiplexed with cyp3a4 expression assays , we performed preliminary imaging staining experiments in heparg , using a variety of cell function and cell tracing reagents relevant to hepatotoxicity ( figure 9 ) . figure 9(a ) shows a phase contrast image of live heparg cells . as image analysis algorithms for phase contrast and bright field images improve and time - lapse imaging becomes more accessible , quantitative , label - free cell health assays will likely see an increase in usage . figure 9(b ) shows mitotracker green fm staining in live heparg cells ; this dye localizes to mitochondria regardless of mitochondrial membrane potential and can be used to determine changes in mitochondrial mass . figure 9(c ) shows monochlorobimane ( mbci ) imaging in live heparg cells ; mbci is nonfluorescent but forms a stable , fluorescent adduct with glutathione ( gsh ) . the fluorescent signal recorded over time is directly proportional to the concentration of gsh and serves as a useful indicator of oxidative stress . figure 9(d ) shows celltracker red cmtpx staining in live heparg cells ; this dye is useful for long - term cell tracing experiments . figure 9(e ) shows live heparg cells stained with tetramethylrhodamine , methyl ester , perchlorate ( tmrm ) ; this dye is widely used to detect changes in mitochondrial membrane permeability in hepatocytes . finally , figure 9(f ) shows alexa fluor 568 phalloidin staining in fixed heparg cells ; this can be used to measure cytoskeletal changes and is widely used as a component of cell viability assays . based on the data presented here , we believe it is now possible , using the culture and assay conditions established in this study , to perform many more multiplexed imaging - based assays using heparg cells , having confidence that metabolic competence is maintained and the hepatocyte subpopulation can be analyzed via quantitative imaging techniques . a plethora of potential multiplexing reagents are available to detect other features of interest , for example , cell death , cell signaling molecules , and functional markers of mechanisms of hepatotoxicity in heparg cells , and hca provides a high - throughput format suitable for large - scale drug and chemical safety screening . looking ahead , more dynamic live cell analysis - based applications for hepatotoxicity seem likely to emerge , perhaps utilizing novel microfluidic devices . we expect that in future hca - compatible live cell substrates for cyp3a4 and other drug metabolism enzymes can be combined with fluorescent biosensors and novel devices for dynamic live hepatocyte cultures in order to shed further light upon the kinetics of drug metabolism and the cellular events underpinning hepatotoxicity . in recent years , powerful automated microscopes and automated image analysis algorithms have become increasingly accessible to more scientists , and these are enabling exciting novel research to be carried out upon individual cells in situ , as opposed to population - based analyses of harvested cells . in this study , we have demonstrated the feasibility of an automated imaging - based approach for assessing cyp3a4 expression and activity using metabolically competent heparg cells . this study has ( a ) demonstrated that cryopreserved differentiated heparg cells retain key functional hepatocyte characteristics , notably maintaining inducible cyp3a4 activity over time , ( b ) shown that these cells may be employed for multiparametric high content analysis applications , and ( c ) provided information on optimal cell culture and image analysis conditions for the application of these cells to imaging - based cyp3a4 expression studies and multiparametric hepatotoxicity assessment . interest in imaging - based assessment of cellular toxicity continues to grow , and hepatotoxicity is of particular interest because of its implications for the pharmaceutical industry . viable alternative cellular models to primary human hepatocytes and nonrepresentative hepatoma cell lines like hepg2 have long been sought . in this study , we have demonstrated that the cryopreserved differentiated form of heparg retains many functional features of human hepatocytes and is highly amenable to quantitative imaging - based assessment of hepatocyte - specific endpoints under conditions where strong and stable cyp3a4 activity is maintained . as imaging - based hepatotoxicity assessment applications continue to emerge , cryopreserved differentiated heparg cells will likely be increasingly employed as metabolically competent surrogates for primary human hepatocytes .

whole - genome sequencing offers unprecedented opportunity to reveal the genetic determinants of phenotypical characteristics of species . the availability of an increasing number of genome sequences allows conducting genome - wide scans of selection aimed at detecting the molecular footprints of adaptation ( heliconius genome consortium 2012 ; qiu et al . 2013 ; zhang et al . 2013 ; fang , nevo , et al . 2014 ; fang , seim , et al . 2014 ; yim et al . , this is often achieved by searching for lineage - specific amino acid changes that might cause functional changes in proteins encoded by candidate genes for a particular trait ( e.g. , zhao et al . however , such a strategy can easily be misleading when only a limited number of sequences are used in the comparisons ( liu 2014 ) . 2011 ) provides a good illustration of both the utility and the potential drawbacks of this approach . the rationale behind sequencing the genome of the naked mole - rat ( heterocephalus glaber ) was the uniqueness of this eusocial rodent species with exceptional longevity , which serves as a model for aging , cancer , and pain resistance ( gorbunova et al . mole - rats have actually evolved convergently in two distinct rodent clades with african mole - rats ( bathyergidae ) belonging to ctenohystrica and spalacids ( spalacidae ) that are part of the mouse - related clade ( blanga - kanfi et al . the genomes of the damaraland mole - rat ( fukomys damarensis ; bathyergidae ) and the blind mole - rat ( nannospalax galili ; spalacidae ) have recently been sequenced to study convergent genomic evolution in these ecologically and phenotypically similar species ( fang , nevo , et al . 2014 ; fang , seim , et al . the naked mole - rat , which was long considered to be part of bathyergidae , has recently been reclassified in its own family ( heterocephalidae ) to better reflect its ancient divergence from other african mole - rats ( more than 30 ma ) and its distinctive morphology ( patterson and upham 2014 ) . the analysis of the naked mole - rat genome sequence has undoubtedly shed light on a number of genetic characteristics that might relate to its numerous peculiar physiological adaptations ( kim et al . was the finding that a single amino acid substitution was responsible for its unique hairless phenotype . indeed , as its name implies , the naked mole - rat is the only wild rodent species that naturally lacks fur . the authors have linked this hairlessness to the presence of a single amino acid substitution in the nuclear receptor corepressor hair growth associated ( hr ) protein , also called hairless . the hr gene is present as a single - copy ortholog in most currently available mammalian genomes with potential paralogs being only found in the gibbon genome ( ensembl release 78 ) . kim et al . ( 2011 ) reported that the naked mole - rat hr sequence is substantially divergent from the other mammals investigated , and that it possesses a tryptophan ( trp / w ) at position 397 of the hr protein , whereas the other mammalian sequences compared exhibit a cysteine ( cys / c ) . because mutations at this particular codon of the hr gene are known to cause hair loss in mice , rat , and men ( panteleyev et al . 1998 ; thompson 2009 ) , they concluded that this specific amino acid substitution was likely responsible for the naked mole - rat hairless phenotype . the molecular evidence for such a conclusion takes the form of a multiple amino acid sequence alignment corresponding to exon 2 of the hr gene in rodents ( exon 3 in human ) presented as supplementary figure s24c of kim et al . first , the authors chose to include only ten additional mammalian species in their hr comparative alignment even tough 32 orthologs of hr where available at the time in databases such as orthomam v6 ( ranwez et al . , we were able to retrieved 92 mammalian orthologous sequences for this exon ( fig . 1 ) . if the cysteine observed at position 397 of the hr protein is likely to be ancestral in mammals because it is also found in monotremes and marsupials , this amino acid is in fact not conserved across placentals . indeed , both the three camel species ( genus camelus ) and the horse ( equus caballus ) exhibit a phenylalanine ( phe / f ) at this position , resulting from two convergent nonsynonymous g->t substitutions at the second position of the corresponding codon . contrary to what is shown in the alignment of supplementary figure s24c of kim et al . ( 2011 ) , the horse hr protein sequence they used ( ncbi accession number : xp_001490941 ) actually has an f instead of a c at position 397 ( supplementary fig . in fact , we verified that all available hr horse sequences have an f at this position ( fig . the donkey ( equus asinus ) also shows an f at this position whereas the rhino , which is the only other available perissodactyl representative , has the ancestral cysteine . we thus come to the conclusion that this position has been mistakenly edited in the alignment of kim et al . 1.maximum likelihood tree obtained from the concatenation of hr exons 1 and 2 for 99 mammals and partial amino acid alignment of exon 2 including position 397 ( boxed ) . the three amino acid substitutions inferred at site 397 are mapped on the corresponding branches of the phylogenetic tree . species are colored according to the placental clades to which they belong : xenarthra ( red ) , afrotheria ( green ) , laurasiatheria ( orange ) , primates ( blue ) , scandentia ( brown ) , glires ( purple ) , and ctenohystrica ( pink ) . note that the ancestral therian and placental branches have been reduced by a factor ten to improve visibility of placental relationships . sequence logos and the 50% majority - rule consensus are indicated as displayed in geneious . 2.partial multiple sequence alignment of hr exon 2 codons and corresponding amino acid translation for 11 perissodactyls including a diversity of extant and extinct equids . maximum likelihood tree obtained from the concatenation of hr exons 1 and 2 for 99 mammals and partial amino acid alignment of exon 2 including position 397 ( boxed ) . the three amino acid substitutions inferred at site 397 are mapped on the corresponding branches of the phylogenetic tree . species are colored according to the placental clades to which they belong : xenarthra ( red ) , afrotheria ( green ) , laurasiatheria ( orange ) , primates ( blue ) , scandentia ( brown ) , glires ( purple ) , and ctenohystrica ( pink ) . note that the ancestral therian and placental branches have been reduced by a factor ten to improve visibility of placental relationships . sequence logos and the 50% majority - rule consensus are indicated as displayed in geneious . partial multiple sequence alignment of hr exon 2 codons and corresponding amino acid translation for 11 perissodactyls including a diversity of extant and extinct equids . second , the guinea pig ( cavia porcellus ) , the closest relative of the naked mole - rat for which annotated genomic data were available at the time of the naked mole - rat genome publication , was not included in the hr protein sequence comparison reported by kim et al . the absence of the guinea pig hr sequence in their alignment is surprising given that sequences of this species have been included in analyses of other 42,399 candidate genes in the same study ( e.g. , terf1 in their supplementary fig . as in the naked mole - rat , the guinea pig hr sequence also has a w at position 397 ( fig . this suggests that this particular amino acid substitution is a shared characteristic of cavia and heterocephalus that both belong to ctenohystrica , which includes ctenodactylids ( gundis ) and hystricognaths ( e.g. , cavia ) ( blanga - kanfi et al . 2009 ) . by sequencing exons 1 and 2 of the hr gene ( corresponding to exons 2 an 3 in human ) in additional ctenohystrican representatives , we demonstrate that the presence of a w at position 397 is in fact a synapomorphy for hystricognaths ( fig . the c397w substitution , which results from a nonsynonymous c->g nucleotide substitution at the third codon position ( fig . 3 ) , most likely occurred in the ancestral lineage of hystricognaths because the w is shared by all representative species investigated , whereas their closest gundi relative ( ctenodactylus vali ) possesses the ancestral cysteine . the closely related damaraland mole - rat ( f. damarensis ; bathyergidae ) , which is fully furred , also arbors the w found in the naked mole - rat . conversely , all available nonhystricognath rodents , including the convergently evolved blind mole - rat ( n. galili ; spalacidae ) , have the ancestral cysteine . because all these rodent species have fur , we conclude that this particular substitution in hr does not explain the hairless phenotype of the naked mole - rat . among the 99 sequences investigated , the ancestral cysteine residue is indeed fixed in the vast majority , including mammalian species with reduced pilosity such as aquatic cetaceans ( chen et al . 2013 ) , the manatee ( trichechus manatus ) , and the walrus ( odobenus rosmarus ) , but also terrestrial species such as the nine - banded armadillo ( dasypus novemcinctus ) , the white rhino ( ceratotherium simum ) , and the african elephant ( loxodonta africana ) . overall , we found no statistical correlation between amino acid changes at position 397 of hr and reduced pilosity across the mammalian phylogeny ( d = 0.0167 , p = 0.68 ; m = 0.0028 , p = 0.79 ) . 3.partial multiple sequence alignment of hr exon 2 codon and corresponding amino acid translation for 25 glires including seven new caviomorph sequences ( * ) . partial multiple sequence alignment of hr exon 2 codon and corresponding amino acid translation for 25 glires including seven new caviomorph sequences ( * ) . we also investigated whether the naked mole - rat hr gene sequence is really divergent from other mammals as suggested by kim et al . based on branch lengths of the maximum likelihood tree obtained from the concatenation of the two exons at the nucleotide level ( fig . 1 ) , the naked mole - rat does not appear to be especially fast evolving compare to other rodents . the hr gene evolves at sensibly the same pace in the two african mole - rats ( heterocephalus and fukomys ) and the convergently evolved spalacid ( nannospalax ) . the apparent sequence divergence of the naked mole - rat hr sequence observed by kim et al . moreover , under the hypothesis that the hr gene is really involved in the naked mole - rat hairless phenotype , it should be possible to detect changes in selective constraints in this particular species . however , the hr gene was not identified as positively selected in recent genome - wide analyses of selection comparing heterocephalus and fukomys ( fang , seim , et al . this case study illustrates the pitfalls of the systematic quest for molecular adaptation by simply looking at point amino acid changes in candidate proteins coupled with the use of only few genome sequences . we suspect that numerous similar cases of false positive results will be revealed once a greater taxonomic diversity of genomes becomes available , as already shown by liu ( 2014 ) for the p53 gene . in mammals , for which a large number of genome sequences are already available , there is no reason not to use this phylogenetic diversity for empowering comparative evolutionary analyses aimed at detecting molecular adaptation . our results also expose the more general problem of the dilution of essential information and figures in the ever - growing supplementary material of genome papers , which are often published in high profile journals where space is reduced . as shown here , such a practice significantly increases the probability that misleading results will escape the scrutiny of editors , reviewers , and ultimately readers . rodent tissue samples came from the mammalian tissue collection of the institut des sciences de levolution de montpellier ( catzeflis 1991 ) . we selected the following ctenohystrica representatives : abrocoma cinerea ( t-0476 ) , c. porcellus ( t-6107 ) , c. vali ( t-0374 ) , ctenomys haigi ( t-3537 ) , cuniculus taczanowskii ( t-3400 ) , dasyprocta leporina ( t-5754 ) , and myoprocta pratti ( t-3541 ) . dna extractions were performed from tissues preserved in 95% ethanol using the dneasy blood & tissue kit ( qiagen ) following manufacturer s instructions . we then polymerase chain reaction ( pcr ) targeted a 562 bp fragment of rodent hr exon 1 using the primer pair hr_ex2hysf ( 5-gcccagcttcctgaaggacac-3)/hr_ex2hysr ( 5-cttgctgcctaggcygaaggc-3 ) and a 584 bp fragment of exon 2 with primer pair hr_ex3hysf ( 5-ctcaggctggcaaaggagcc-3)/hr_ex3hysr ( 5-ctgcctgcyctcttcaggg-3 ) . the following pcr conditions were used for the two exons : 95 c for 4 min ( initial denaturation ) , followed by 30 cycles at 95 c for 20 s ( denaturation ) , 58 c ( exon 1 ) and 60 c ( exon 2 ) for 30 s ( hybridization ) , 72 c for 30 s ( extension ) , and a final extension step at 72 c for 10 min . purified amplicons were sanger sequenced on both strands using the pcr primers with the big dye terminator v3.1 kit ( applied biosystem ) on an applied abi prism 3130xl automated sequencer . the newly obtained sequences have been deposited in the european nucleotide archive under accession numbers ln680723ln680736 . complete mammalian hr cdss and hr exons 1 and 2 were extracted from the orthomam v8 database ( douzery et al . these alignments were subsequently enriched by annotated sequences harvested from ongoing mammalian genome projects available as whole genome shotgun assemblies in genbank . newly obtained sequences for hr exons 1 and 2 were added to available orthologous sequences leading to a total of 99 sequences . concatenations of hr exons 1 and 2 were built for 99 mammals and 25 species of glires , respectively . ambiguously aligned codons were then excluded using the gblocks server ( castresana 2000 ) with default relaxed parameters . maximum likelihood phylogenetic trees were inferred from nucleotide sequences for the exon data sets using the phyml 3.0 ( guindon et al . 2009 ) plugin of geneious r7 ( kearse et al . 2012 ) under the gtr+g8 model using spr branch swapping on a bionj starting tree and 100 booststrap replications . bayesian phylogenetic inference under a mixed model was conducted using the mpi version of mrbayes 3.2.3 ( ronquist et al . two independent runs of four incrementally heated mcmcmc starting from a random tree were performed . mcmcmc were run for 1,000,000 generations with trees and associated model parameters being sampled every 1,000 generations . the initial 250 trees in each run were discarded as burn - in samples after convergence checking . the 50% majority - rule bayesian consensus tree and the associated posterior probabilities were computed from the 1,500 combined trees sampled in the two independent runs . the correlation between amino acid changes at position 397 of hr and reduced pilosity was tested by calculating the d and m association statistics ( huelsenbeck et al . 2003 ) as implemented in simmap 1.5 ( bollback 2006 ) . to account for phylogenetic uncertainty , we used 15 trees subsampled from the previous posterior sample of 1,500 trees obtained with mrbayes . amino acid changes were recoded as a multistate character ( c = 0 ; w = 1 ; f = 2 ) and pilosity was coded as a binary character ( 0 = fully furred ; 1 = reduced pilosity ; see fig .

asd is a group of increasingly recognised lifespan persistent neurodevelopmental disorders of early onset [ 3 , 4 ] characterised by abnormalities in language , social interaction and a range of stereotyped and repetitive behaviours . however , autism ( the paradigmatic asd ) is both highly heritable , and associated with alterations in both pre - natal brain development and post - natal brain structure [ 8 , 9 ] . despite this , little is known regarding the influence of specific genes on brain anatomy in asd with the exception of the serotonin transporter and monoamine oxidase genes which contain functional genetic variants that have been associated with cortical volume in children with asd . a strong and as yet unexamined candidate gene for modulating brain anatomy in asd is brain derived neurotrophic factor ( bdnf ) which plays a key role in neurogenesis , cortical lamination , synaptic plasticity , and neuron survival . intracellular packaging and secretion of bdnf is altered by a common functional [ 13 , 14 ] non - synonymous single nucleotide polymorphism ( snp ) within the 5 region of the gene ( bdnf val66met ) which codes for a valine to methionine substitution . in the healthy population , genotype at this polymorphism has been associated with differences in cortical plasticity and regional cortical volume ( cv ) [ 1621 ] ( see table 1 ) . many of the cortical areas where bdnf val66met genotype influences cv in typically developing individuals overlap with those where structural differences have been reported between people with asd and typically developing controls . table 1studies in healthy controls relating bdnf val66met to corticalfirst authoryearsample sizeage ( years)gendermeasurefindingsvalvalmetpezawas20046942 m : 34 r : 18 - 60mixedvbmmet carriers - reduced cv in bilateral inferior and middle frontal gyri , left precuneus and superior parietal lobule , right medial frontal , precentral , superior frontal , postcentral gyri . val homozygotes - reduced cv in right middle frontal and lingual gyri.agartz20066630m : 42mixedautomated lobar cvno significant effect of genotypenemoto20064168 m : 36 r : 20 - 72mixedvbmmet carriers - reduced cv in left parahippocampal gyrus.varnas20087331m : 42mixedcortical thicknesstrend towards association between genotype and cv in left frontal ( straight gyrus , transverse frontopolar , superior frontal ) and right frontal ( pars triangularis ) cortices . no effect of genotype on regional ctschofield20089659 m : 32 r : 20 - 60mixedvbmno effect of genotype in regional cvabbreviations : m mean , r range , vbm voxel based morphometry , cv cortical volume , ct cortical thickness studies in healthy controls relating bdnf val66met to cortical abbreviations : m mean , r range , vbm voxel based morphometry , cv cortical volume , ct cortical thickness for example three independent studies [ 16 , 17 , 19 ] have reported regional cortical cv reductions in bdnf met carriers compared to val homozygotes in regions such as the dorso - lateral and inferior frontal , superior parietal and precuneus cortices . these are cortical regions where our group , and others that have shown structural differences between people with asd and healthy controls . furthermore , all [ 2428 ] but one of the studies examining bdnf measures in asd report abnormalities . autism spectrum disorder has been associated with ( i ) elevated serum bdnf in infancy childhood and adulthood [ 25 , 26 ] , ( ii ) elevated blood lymphocyte bdnf mrna in young adults , and ( iii ) increased basal forebrain bdnf levels in post - mortem samples of cerebral cortex . therefore , it is possible that altered bdnf signalling in asd contributes to the anatomical differences of people with asd by disturbing the pathways through which the bdnf val66met polymorphism influences regional cv . it is not known however if the cv correlates of bdnf val66met genotype differ between people with asd and typically developing controls . therefore , we carried out a preliminary exploratory investigation in which we related bdnf val66met genotype to 33 regional measures of cv in 71 individuals 41 of whom had an asd diagnosis ( 35 with asperger syndrome , four with high - functioning autism and two with other pervasive developmental disorders ) , and 30 typically developing controls . furthermore , in those regions where there were significant differences between people with asd and controls in the relationship between bdnf val66met genotype and cv we also examined if these were accompanied by alterations in cortical thickness ( ct ) or surface area ( sa ) . the volume of a cortical region is approximated by the product of the total surface area and the mean cortical thickness of that region . because these two sole determinants of cv reflect distinct evolutionary and developmental processes [ 31 , 32 ] it is important to disambiguate their relative contribution to any differences in the cv correlates of bdnf val66met genotype in people with asd and controls . for example , sa is influenced by division of progenitor cells in the embryological periventricular area and varies as a function of brain volume and cortical folding ( gyrification ) . in contrast , ct is likely to reflect dendritic arborization / pruning within the grey matter or myelination at the grey / white matter interface . were recruited through a hospital - based national clinic and a university department specialising in the assessment of asd with infrastructure support from the medical research council united kingdom autism imaging multicentre study ( mrc uk a.i.m.s ) program . all cases were diagnosed clinically with asperger syndrome ( n = 35 ) , autism ( n = 4 ) , or pervasive - developmental disorder - not otherwise specified ( n = 2 ) according to dsm - iv criteria . diagnoses were made by a team of senior clinicians trained in the autism diagnostic interview - revised ( adi - r ) ( le couteur et al . 1989 ) and the autism diagnostic observation schedule ( ados - g ) ( lord et al . twenty - four of the participants with asd also agreed to confirmation of clinical diagnosis using the adi - r or ados . typically developing healthy controls participants in the study underwent structured physical and psychiatric examination ( for the presence of a co - morbid dsm - iv axis i or ii disorder ) . participants did not have a history of physical or psychiatric disorder affecting brain function ( e.g. , epilepsy or psychosis ) , or a genetic disorder putatively associated with autistic spectrum disorders ( e.g. , fragile x syndrome ) . all participants had full - scale intelligence quotients above 70 . all subjects ( or guardians where applicable ) gave informed and written consent . there were no statistically significant differences in mean age between cases and controls or between bdnf val homozygotes and bdnf met carriers . table 2subject characteristicscharacteristiccasescontrolsnumber4130male3426age at scan in years mean34.6427.5 range12641251standard deviation12.5511.89fsiq mean104.58126.5 range7313583155 standard deviation17.2319.09genotype bdnf val val3119 bdnf met carrier1011 subject characteristics the bdnf val66met genotype was determined using a taqman allele specific assay method ( applied biosystems ) according to the manufacturer s protocols . polymerase chain reaction ( pcr ) amplifications were performed on an abi prism 7000 sequence detection system ( applied biosystems ) with the reaction mixture in a total volume of 2 l , consisting of 5 ng of genomic dna , 2 universal pcr master mix ( abgene ) , 40 taqman snp genotyping assay mix ( applied biosystems ) , and deionized h2o . after denaturing at 95c for 15 min , 50 cycles of pcr were performed under the following conditions : 92c for 15 s and 60c for 90 s. all genotypes were reported with the allelic discrimination program using the applied biosystems sequence detection system ( sds ) . brain mris were acquired using a ge signa 1.5 t neuro - optimised mr system ( general electric , milwaukee wi , usa ) . freesurfer freeware ( http://surfer.nmr.mgh.harvard.edu/fswiki ) was used to derive models of the cortical sheet in each t1-weighted image and parcellate out the cortex into 33 regions . these well - validated [ 35 , 36 ] and fully automated procedures have been extensively described elsewhere [ 37 , 38 ] , and we will only provide a brief description here . this involved intensity normalisation , the removal of extra - cerebral tissues using a skull stripping algorithm , and image segmentation using a connected components algorithm . the output of this stage is a single filled white - matter volume for each cerebral hemisphere . a surface tessellation was then generated for each white- matter volume by fitting a deformable template . the grey matter / csf surface was also modelled using a similar process . given explicit models for the white / grey and grey / csf surfaces , the measure of absolute cortical thickness at any given point on the white / grey matter surface is then taken to be the shortest distance between that point and the grey / csf surface . this measurement is made at approximately 150,000 points across each hemisphere for each scan . in order to achieve automated parcellation of each individual cortical hemispheric sheet into 33 regions , a probabilistic atlas was placed within a surface - based co - ordinate system to which each scan was aligned . parcellation generated a measure of total cv , total sa and average ct for each sub - region within each hemisphere . for each sub - region values for left and right hemispheres were combined , resulting in a total of three measures ( sa , ct and gv ) for each of 33 sub - regions . analyses of covariance ( ancovas ) were used to model the effects of group ( asd vs controls ) , bdnf genotype ( val vs met carriers ) and the interaction of these two terms on regional measures of cv . in order to limit the number of statistical tests , we combined measures of cv for each of 33 cortical regions per hemisphere into total bilateral cv for that region . given the exploratory nature of this analysis we adopted an uncorrected p value of 0.05 . interaction effects , we carried out further ancova s for ct and sa in order to establish which of these parameters may be contributing to group - by - genotype cortical volume , sa and ct were not transformed before being used as dependent variables in these analyses . within this cross - sectional dataset , we were not able to identify the non - linear age effects reported in much larger , longitudinal studies . linear age effect were however seen and given the wide age - range of our sample , age was included as a co - variate to remove variance not related to our independent variables of interest . as there were no significant differences between groups in gender , or intracranial volume ( icv ) we did not co - vary for these in analyses . furthermore , the relationships between icv and cv , sa / gyrification and ct are complex , non - linear and may differ with age and gender . were recruited through a hospital - based national clinic and a university department specialising in the assessment of asd with infrastructure support from the medical research council united kingdom autism imaging multicentre study ( mrc uk a.i.m.s ) program . all cases were diagnosed clinically with asperger syndrome ( n = 35 ) , autism ( n = 4 ) , or pervasive - developmental disorder - not otherwise specified ( n = 2 ) according to dsm - iv criteria . diagnoses were made by a team of senior clinicians trained in the autism diagnostic interview - revised ( adi - r ) ( le couteur et al . 1989 ) and the autism diagnostic observation schedule ( ados - g ) ( lord et al . twenty - four of the participants with asd also agreed to confirmation of clinical diagnosis using the adi - r or ados . typically developing healthy controls participants in the study underwent structured physical and psychiatric examination ( for the presence of a co - morbid dsm - iv axis i or ii disorder ) . participants did not have a history of physical or psychiatric disorder affecting brain function ( e.g. , epilepsy or psychosis ) , or a genetic disorder putatively associated with autistic spectrum disorders ( e.g. , fragile x syndrome ) . all participants had full - scale intelligence quotients above 70 . all subjects ( or guardians where applicable ) gave informed and written consent . there were no statistically significant differences in mean age between cases and controls or between bdnf val homozygotes and bdnf met carriers . table 2subject characteristicscharacteristiccasescontrolsnumber4130male3426age at scan in years mean34.6427.5 range12641251standard deviation12.5511.89fsiq mean104.58126.5 range7313583155 standard deviation17.2319.09genotype bdnf val val3119 bdnf met carrier1011 subject characteristics the bdnf val66met genotype was determined using a taqman allele specific assay method ( applied biosystems ) according to the manufacturer s protocols . polymerase chain reaction ( pcr ) amplifications were performed on an abi prism 7000 sequence detection system ( applied biosystems ) with the reaction mixture in a total volume of 2 l , consisting of 5 ng of genomic dna , 2 universal pcr master mix ( abgene ) , 40 taqman snp genotyping assay mix ( applied biosystems ) , and deionized h2o . after denaturing at 95c for 15 min , 50 cycles of pcr were performed under the following conditions : 92c for 15 s and 60c for 90 s. all genotypes were reported with the allelic discrimination program using the applied biosystems sequence detection system ( sds ) . brain mris were acquired using a ge signa 1.5 t neuro - optimised mr system ( general electric , milwaukee wi , usa ) . freesurfer freeware ( http://surfer.nmr.mgh.harvard.edu/fswiki ) was used to derive models of the cortical sheet in each t1-weighted image and parcellate out the cortex into 33 regions . these well - validated [ 35 , 36 ] and fully automated procedures have been extensively described elsewhere [ 37 , 38 ] , and we will only provide a brief description here . this involved intensity normalisation , the removal of extra - cerebral tissues using a skull stripping algorithm , and image segmentation using a connected components algorithm . the output of this stage is a single filled white - matter volume for each cerebral hemisphere . a surface tessellation was then generated for each white- matter volume by fitting a deformable template . the grey matter / csf surface was also modelled using a similar process . given explicit models for the white / grey and grey / csf surfaces , the measure of absolute cortical thickness at any given point on the white / grey matter surface is then taken to be the shortest distance between that point and the grey / csf surface . this measurement is made at approximately 150,000 points across each hemisphere for each scan . in order to achieve automated parcellation of each individual cortical hemispheric sheet into 33 regions , a probabilistic atlas was placed within a surface - based co - ordinate system to which each scan was aligned . parcellation generated a measure of total cv , total sa and average ct for each sub - region within each hemisphere . for each sub - region values for left and right hemispheres were combined , resulting in a total of three measures ( sa , ct and gv ) for each of 33 sub - regions . analyses of covariance ( ancovas ) were used to model the effects of group ( asd vs controls ) , bdnf genotype ( val vs met carriers ) and the interaction of these two terms on regional measures of cv . in order to limit the number of statistical tests , we combined measures of cv for each of 33 cortical regions per hemisphere into total bilateral cv for that region . given the exploratory nature of this analysis we adopted an uncorrected p value of 0.05 . interaction effects , we carried out further ancova s for ct and sa in order to establish which of these parameters may be contributing to group - by - genotype effects for cv ( again , applying an uncorrected p value of 0.05 ) . cortical volume , sa and ct were not transformed before being used as dependent variables in these analyses . within this cross - sectional dataset , we were not able to identify the non - linear age effects reported in much larger , longitudinal studies . linear age effect were however seen and given the wide age - range of our sample , age was included as a co - variate to remove variance not related to our independent variables of interest . as there were no significant differences between groups in gender , or intracranial volume ( icv ) we did not co - vary for these in analyses . furthermore , the relationships between icv and cv , sa / gyrification and ct are complex , non - linear and may differ with age and gender . neither group , genotype , not the interaction of these two terms was associated with total intracranial volume , total cortical volume , total cortical surface area or mean cortical thickness . interaction for cv in six cortical areas - posterior cingulate , caudal anterior cingulate , rostral middle frontal , lateral orbitofrontal , pars orbitalis and pars triangularis . further , within all of these regions , significant group by genotype interactions were also found for sa , but not ct ( table 3 ) . in order to determine how specific group - by - genotype interactions for sa were to those regions also showing interactions for cv , we next ran ancovas for sa in all cortical regions . table 3results for analyses of covariance for regional cortical measuresregionmeasurevolumesurface areathicknessgroupbdnfg x bgroupbdnfg x bgroupbdnfg x bfpfpfpfpfpfpfpfpfpbanksts4.820.031.990.161.590.21caudantcing0.020.890.610.446.310.010.110.740.460.508.440.0050.000.950.000.990.150.70caudmidfr4.150.051.030.310.730.40cuneus0.270.600.020.900.140.71entorhinal0.300.590.270.600.010.91fusiform2.970.090.180.670.920.34infpar4.120.052.390.130.060.81inftemp0.280.602.350.131.990.16isthcing2.530.124.470.043.340.07latoccip5.040.031.010.320.430.51latorbfr0.500.480.230.635.250.030.490.490.230.635.250.030.510.480.270.610.160.69lingual1.190.280.240.630.140.71medorbfr0.030.860.630.431.730.19midtemp1.840.180.220.640.840.36parahipp0.500.480.920.344.230.040.060.940.680.412.470.120.720.400.640.430.780.38paracentral3.770.062.760.101.270.27parsoperc0.800.370.720.401.050.31parsorbit0.660.422.580.114.770.030.140.711.320.254.360.041.290.261.190.280.780.38parstriang0.080.930.070.804.170.050.000.990.010.925.460.020.120.760.010.940.050.83pericalc1.410.240.150.700.460.50postcentral0.970.330.000.990.020.90postcing0.520.480.560.464.160.050.280.600.280.607.400.0080.900.370.590.440.350.56precentral3.060.090.420.520.00.35precuneus3.480.071.450.232.330.13rostrantcing0.200.661.400.240.270.61rostrmidfr0.180.670.030.874.300.040.010.910.050.825.300.020.870.360.000.990.340.56supfr1.230.271.820.180.890.35suppar0.200.660.080.791.650.20suptemp0.170.680.580.450.700.41supramarg0.260.623.330.070.390.54frontalpole0.030.870.140.710.070.80temppole0.070.790.820.390.510.48transvtemp0.660.421.840.181.110.30 results for analyses of covariance for regional cortical measures in all six regions group by genotype interactions for both cv and sa reflected the same phenomenon lower cv / sa in control bdnf met allele carriers compared to control bdnf val homozygotes , but higher cv / sa in asd bdnf met allele carriers as compared to asd bdnf val homozygotes . the same pattern was seen in all six regions and is illustrated in fig . bars represent 95% confidence intervals for mean cv / sa caudal anterior and posterior cingulate cv and sa by group and bdnf genotype . the results from our exploratory study suggest that the relationship between cortical anatomy and bdnf genotype may differ between people with asd and typically developing controls . given the preliminary nature of our investigation we did not correct for multiple comparisons , and so replication is required . firstly , rather than being generalised , group - by - genotype interactions may be most marked in particular cortical regions , i.e the caudal anterior cingulate , posterior cingulate , rostral middle frontal , lateral orbitofrontal , pars orbitalis and pars triangularis . these regions are not scattered across the cortex , but describe two larger contiguous frontal areas secondly , many of the cortical regions where we identified group - by - genotype interactions for cv / sa overlap with those where bdnf has been shown to modulate cv in typically developing by structural mri [ eg in the medial , middle and inferior frontal [ 16 , 17 , 19 ] ] , and n - acetylaspartate levels ( a marker of neuronal integrity ) in healthy controls by magnetic resonance spectroscopy [ anterior cingulate ] . in our sample the relationship between cv and bdnf val66met genotype in controls was in the same direction and similar frontal distribution as that reported in other samples of typically developing individuals [ 16 , 17 ] . that is in controls cv was reduced in bdnf met allele carriers compared to bdnf val allele homozygotes . furthermore , cortical regions where we identified group - by - genotype interactions for cv / sa also overlap with those where compared to controls , people with asd have been reported as showing alterations in cv , cortical folding and magnetic resonance spectroscopy measures of neuronal integrity [ anterior cingulate ] . this suggests that differences in cortical structure between people with asd and typically developing controls may be due to differences in bdnf val66met genotype frequency and/or function . thirdly , in those ( and only those ) regions showing significant group x genotype interactions for cv , we also found significant group x genotype the sa within a given cortical region may be altered in three ways ; ( i ) alterations in radial brain size , ( ii ) displacement of the landmarks ( eg sulcal landmarks ) used to define regional boundaries , or ( ii ) increased gyrification of the cortical sheet within unaltered regional boundaries . interactions for intracranial volume or total cerebral sa ( data not shown but available ) , which argues against the first of these explanations . the remaining two explanations are compatible with existing reports of altered sulcal positioning and gyrification in people with asd compared to controls . as both of these cortical characteristics can be quantified from mri scans their relative contributions to putative differences in the sa correlates of bdnf val66met genotype between people with asd and controls can be directly examined in future work . the mechanisms that could underlie differences between people with asd and controls in the cv / sa correlates of bdnf val66met genotype are unclear . such differences could be due to abnormal bdnf signalling in asd causing disruption of the pathways through which the bdnf val66met polymorphism might influence cortical structure during typical development such as neuronal migration and/or experience dependent plasticity . alternatively , our findings may not be due to abnormal bdnf signalling in asd per se , but reflect the fact that brain structure is so atypical in asd that the subtle differences accounted for by any given single genetic polymorphisms are drowned out a further possibility is that due to dysfunction within molecular pathways that interact with bdnf to influence brain development the typical downstream effects of differences in bdnf signalling due to val66met genotype are not seen in asd . autism spectrum disorder has been associated with reports of abnormalities within several discrete molecular systems that all share the feature of directly impacting on or interacting with bdnf transcription and function . these include serotonin ( 5ht ) [ 50 , 51 ] , calcium - dependent activator protein for secretion 2 ( caps2 ) [ 52 , 53 ] , methyl - cpg binding protein 2 ( mecp2 ) , and jumonji at - rich interactive domain 1c ( jarid1c ) . our study does however represent the first examination of the relationship between bdnf and brain anatomy in asd , and is equivalent in sample size to some of the smaller studies that have been published to date on bdnf vall66met and brain structure in typically developing controls [ 55 , 56 ] . secondly we were not able to obtain adi - r / ados confirmation of clinical asd diagnosis in all volunteers in our asd group . however , this confirmation was available for the majority of people with asd , and all asd diagnoses were made by a multidisciplinary team trained in the adi - r and ados . thirdly , almost all participants with asd in our study had a diagnosis of asperger syndrome , and all had a fsiq greater than 70 . further studies in diagnostically homogeneous samples of people with other asd diagnoses and cognitive profiles will be required to establish the generalisability of our findings across the autism spectrum . although this means that our findings may be driven my differences in fsiq rather than asd diagnosis per se , the strong association between asd and learning disability complicates the issue of matching for iq in study design . furthermore , in our sample , fsiq did not correlate with any of the regional cortical measures showing group - by - genotype interactions . fifthly , because our sample covered a wide age - range , we co - varied for age in all analyses and were not therefore able to directly model possible group - by - genotype - by - age the age range of our sample and the predominance of individuals with asperger syndrome diagnoses may also account for the fact that we did not observe the increased cortical volumes commonly reported in autism [ 58 , 59 ] . finally , as our sample included both males and females we were not able to determine the influence of gender on our findings . in summary , our results provide preliminary evidence that in medial and lateral frontal cortices the relationship between bdnf val66met genotype and cortical volume is not the same in people with asd as it is in typically developing populations . this may be due to differences between people with asd and typically developing populations in either ( i ) bdnf val66met genotype frequencies , or ( ii ) the mechanisms linking this genetic polymorphism to regional variations in cortical surface area .

peritonitis carcinomatosa indicates the presence of malignant cells in the peritoneal cavity , and is a well known complication of a number of malignant diseases . as a result , so - called malignant ascites develops . malignant ascites is characterized by positive cytology of malignant cells . impaired lymphatic drainage by occlusion of the lymphatic vessels and increased fluid production causes the accumulation of malignant ascites in the peritoneum.1 clinical manifestations include symptoms of abdominal pain , obstruction , fatigue , and abdominal swelling . in their retrospective analysis of 209 patients with malignant ascites , ayantunde et al found a median survival time of 5.7 months after diagnosis of ascites . independent negative prognostic factors were type of cancer , liver metastasis , and low serum albumin , and , in contrast , patients with ovarian cancer had a favorable prognosis.2 treatment options are symptomatic , and the most common is paracentesis or surgical treatment , including peritoneovenous shunting . systemic and local treatment options include systemic chemotherapy , intraperitoneal chemotherapy , or therapy with radioisotopes . additionally , the vascular endothelial growth factor antibody bevacizumab is currently being tested in clinical studies to treat and prevent malignant ascites.3 catumaxomab ( anti - epcam anti - cd3 ) is a hybrid , hybridoma - derived , trifunctional , monoclonal bispecific antibody , combining two half - antibodies of mouse igg2a and rat igg2b that represent homologous immunoglobulin subclasses . preclinical studies have shown the following three events to occur , demonstrating the trifunctional mode of action of the drug : one antigen binding site , the mouse igg2a , recognizes the tumor - specific antigen4the other antigen binding site ( rat igg2b ) binds to cd3 , part of the t cell receptor complex5,6the fc - fragment binds to fcr type i and iii positive cells ( eg , macrophages , dendritic cells , and natural killer cells ) but not to inhibitory type ii fcr ( located on , eg , b cells).7 one antigen binding site , the mouse igg2a , recognizes the tumor - specific antigen4 the other antigen binding site ( rat igg2b ) binds to cd3 , part of the t cell receptor complex5,6 the fc - fragment binds to fcr type i and iii positive cells ( eg , macrophages , dendritic cells , and natural killer cells ) but not to inhibitory type ii fcr ( located on , eg , b cells).7 the stimulation of immune cells is demonstrated by production of cytokines , including interleukin ( il)-1 , il-2 , il-6 , il-12 , and the dendritic cell cytokine 1 . activated accessory cells , including macrophages and dendritic cells , induce several costimulatory signals , eg , via cd40-cd40l to t cells to prevent t cell anergy . the simultaneous activation of t cells and accessory immune cells , and their mutual stimulation , leads to specific tumor cell killing by induction of apoptosis , release of cytokines , and perforin - mediated lysis , as well as antibody - dependent cellular cytotoxicity . crucial in this process is the combination of the two potent immunoglobulin isotypes , mouse igg2a and rat igg2b , which , in contrast with other reported combinations , not only bind but also activate accessory cells . the trifunctional approach thus leads to major histocompatibility complex - unrestricted but specific killing of tumor cells without need for preactivation or external costimulation5,8 ( see figure 1 ) . epcam is a type i transmembrane glycoprotein that mediates ca - independent homophilic cell adhesions.9 in humans , epcam is expressed only in epithelium and neoplasms derived from epithelia.10 epcam is also strongly expressed in carcinomas of various origins , including colon , rectum , gastric , ovarian , esophagus , lung , pancreas , breast , head , and neck.1113 because of the wide expression on tumor cells , epcam is considered a tumor - associated antigen , and innovative immunotherapeutic approaches targeting epcam are of special interest . epcam expression is a negative prognostic marker for overall survival in patients suffering from either nodal - positive or node - negative breast cancer . schmidt et al demonstrated that epcam overexpression was independently associated with poor survival in node - negative patients . this effect was particularly strong in the subgroup of triple - negative breast cancer , making epcam an attractive therapeutic target in this patient population.14,15 epcam has a direct impact on cell cycle and proliferation , and the ability to upregulate the proto - oncogene , c - myc , and cyclin a / e rapidly . furthermore , epcam weakens cadherin - mediated cell adhesion , and thereby modulates proliferation , differentiation , and tissue maintenance.16 blocking of epcam leads to a decrease in proliferation and metabolism in human carcinoma cells.17 recently , maetzel et al demonstrated how epcam affects nuclear function by shedding of its ectodomain , epex , and nuclear translocation of its intracellular domain , epicd , dependent on the presence of tumor necrosis factor - alpha - converting enzyme ( tace , adam17 ) and presenilin-2.18 furthermore , lindhofer et al showed elimination of putative epcam - positive cancer stem cells ( cd 133+/epcam+ ) in patients with malignant ascites treated with catumaxomab.19 recently hirschhaeuser et al demonstrated a strong , dose - dependent effect of catumaxomab on multicellular tumor spheroids of human epcam - positive fadu tumor cells when cocultured with human peripheral blood monocytes in terms of volume reduction and infiltration of immune cells.20 the first reported clinical pilot study treated eight patients with malignant ascites by intraperitoneal application of catumaxomab or ertumaxomab binding epcam or human epidermal growth receptor 2/neu antigen on tumor cells , respectively . treatment consisted of four to six applications within nine to 23 days , using a total amount of 145940 g of the antibody . seven of eight patients required no further paracentesis during follow - up or until death , with a mean paracentesis - free interval of 38 weeks . complete elimination of tumor cells in ascites was seen at total doses of 40140 g . clinical response with disappearance of ascites accumulation was correlated with elimination of tumor cells ( p = 0.0014).21 in a phase i / ii study reported by burges , patients with malignant ascites due to ovarian cancer were treated with escalating intraperitoneal doses of catumaxomab . the maximum tolerated dose was defined as 10 , 20 , 50 , 200 , and 200 g on days 0 , 3 , 6 , 9 , and 13 . the dose - limiting toxicities were large bowel obstruction common toxicity criteria grade 3 and gamma glutamyl transferase elevation grade 4 . all patients had treatment - emergent adverse events , with fever , nausea , vomiting , abdominal pain , lymphopenia , and general pain being the most common events . in terms of efficacy , 22 of 23 patients did not require any further paracentesis during a follow - up period of up to 37 days . the authors concluded that a dose regimen of 10 , 20 , 50 , and 150 g would be the recommended treatment schedule for further investigation.22 this led to a pivotal phase ii / iii study in patients with symptomatic malignant ascites secondary to epithelial cancers requiring symptomatic therapeutic paracentesis . the study compared paracentesis with intraperitoneal catumaxomab versus paracentesis alone in a two - arm , open - label , randomized trial . the primary endpoint was puncture - free survival , defined as the time to first need for therapeutic puncture or death after treatment . secondary endpoints were time to next paracentesis , ascites signs defined by the patient , ascites signs defined by the investigator , and overall survival . the investigators were to follow an algorithm when a paracentesis was indicated ( ascites > 1l as assessed by a computed tomography scan and signs and symptoms of ascites assessed by the investigator using physical examination and a patient questionnaire ) to ensure a comparable decision on when to perform paracentesis by the different investigators in the different treatment arms . patient in the paracentesis - only treatment group were allowed to cross over to catumaxomab treatment if they still fulfilled the inclusion and exclusion criteria , and had had at least two paracenteses after day 0 of the study . the percentage and outcome of the crossover patients were not reported , although they might influence the secondary endpoint of overall survival . treatment consisted , as recommended , of four constant - rate intraperitoneal infusions at doses of 10 , 20 , 50 , and 150 g of catumaxomab on days 0 , 3 , 7 , and 10 . the antibody was administered via intraperitoneal catheter in an inpatient setting , and the control group was treated with a paracentesis . toxicity was as expected , with predominantly cytokine - release - like symptoms , including pyrexia ( in 60.5% of patients ) , nausea , and vomiting . in total , 258 patients were randomized , of whom 170 received catumaxomab and paracentesis and 88 received paracentesis alone . one hundred and twenty - nine patients had ovarian cancer , and 129 patients suffered from nonovarian cancer , mostly gastric cancer ( n = 66 ) . the primary endpoint of puncture - free survival was significantly prolonged in catumaxomab patients in both strata ( ovarian : 52 versus 11 days , and nonovarian cancer : 37 versus 14 days , p < 0.0001 , respectively ) as well as in the pooled analysis ( 46 versus 11 days ) . the secondary endpoint of median overall survival was not prolonged in the pooled analysis ( 72 days for catumaxomab versus 68 days for paracentesis only , p = 0.0846 ) as well as in the stratified groups . subgroup analysis showed a significant ( p = 0.0313 ) survival benefit for catumaxomab in the gastric cancer patients ( 71 versus 44 days ) . the authors concluded that the treatment regimen demonstrated a clinically relevant benefit in patients with malignant ascites from epithelial cancer.23 catumaxomab ( anti - epcam anti - cd3 ) is a hybrid , hybridoma - derived , trifunctional , monoclonal bispecific antibody , combining two half - antibodies of mouse igg2a and rat igg2b that represent homologous immunoglobulin subclasses . preclinical studies have shown the following three events to occur , demonstrating the trifunctional mode of action of the drug : one antigen binding site , the mouse igg2a , recognizes the tumor - specific antigen4the other antigen binding site ( rat igg2b ) binds to cd3 , part of the t cell receptor complex5,6the fc - fragment binds to fcr type i and iii positive cells ( eg , macrophages , dendritic cells , and natural killer cells ) but not to inhibitory type ii fcr ( located on , eg , b cells).7 one antigen binding site , the mouse igg2a , recognizes the tumor - specific antigen4 the other antigen binding site ( rat igg2b ) binds to cd3 , part of the t cell receptor complex5,6 the fc - fragment binds to fcr type i and iii positive cells ( eg , macrophages , dendritic cells , and natural killer cells ) but not to inhibitory type ii fcr ( located on , eg , b cells).7 the stimulation of immune cells is demonstrated by production of cytokines , including interleukin ( il)-1 , il-2 , il-6 , il-12 , and the dendritic cell cytokine 1 . activated accessory cells , including macrophages and dendritic cells , induce several costimulatory signals , eg , via cd40-cd40l to t cells to prevent t cell anergy . the simultaneous activation of t cells and accessory immune cells , and their mutual stimulation , leads to specific tumor cell killing by induction of apoptosis , release of cytokines , and perforin - mediated lysis , as well as antibody - dependent cellular cytotoxicity . crucial in this process is the combination of the two potent immunoglobulin isotypes , mouse igg2a and rat igg2b , which , in contrast with other reported combinations , not only bind but also activate accessory cells . the trifunctional approach thus leads to major histocompatibility complex - unrestricted but specific killing of tumor cells without need for preactivation or external costimulation5,8 ( see figure 1 ) . epcam is a type i transmembrane glycoprotein that mediates ca - independent homophilic cell adhesions.9 in humans , epcam is expressed only in epithelium and neoplasms derived from epithelia.10 epcam is also strongly expressed in carcinomas of various origins , including colon , rectum , gastric , ovarian , esophagus , lung , pancreas , breast , head , and neck.1113 because of the wide expression on tumor cells , epcam is considered a tumor - associated antigen , and innovative immunotherapeutic approaches targeting epcam are of special interest . epcam expression is a negative prognostic marker for overall survival in patients suffering from either nodal - positive or node - negative breast cancer . schmidt et al demonstrated that epcam overexpression was independently associated with poor survival in node - negative patients . this effect was particularly strong in the subgroup of triple - negative breast cancer , making epcam an attractive therapeutic target in this patient population.14,15 epcam has a direct impact on cell cycle and proliferation , and the ability to upregulate the proto - oncogene , c - myc , and cyclin a / e rapidly . furthermore , epcam weakens cadherin - mediated cell adhesion , and thereby modulates proliferation , differentiation , and tissue maintenance.16 blocking of epcam leads to a decrease in proliferation and metabolism in human carcinoma cells.17 recently , maetzel et al demonstrated how epcam affects nuclear function by shedding of its ectodomain , epex , and nuclear translocation of its intracellular domain , epicd , dependent on the presence of tumor necrosis factor - alpha - converting enzyme ( tace , adam17 ) and presenilin-2.18 furthermore , lindhofer et al showed elimination of putative epcam - positive cancer stem cells ( cd 133+/epcam+ ) in patients with malignant ascites treated with catumaxomab.19 recently hirschhaeuser et al demonstrated a strong , dose - dependent effect of catumaxomab on multicellular tumor spheroids of human epcam - positive fadu tumor cells when cocultured with human peripheral blood monocytes in terms of volume reduction and infiltration of immune cells.20 the first reported clinical pilot study treated eight patients with malignant ascites by intraperitoneal application of catumaxomab or ertumaxomab binding epcam or human epidermal growth receptor 2/neu antigen on tumor cells , respectively . treatment consisted of four to six applications within nine to 23 days , using a total amount of 145940 g of the antibody . seven of eight patients required no further paracentesis during follow - up or until death , with a mean paracentesis - free interval of 38 weeks . complete elimination of tumor cells in ascites was seen at total doses of 40140 g . clinical response with disappearance of ascites accumulation was correlated with elimination of tumor cells ( p = 0.0014).21 in a phase i / ii study reported by burges , patients with malignant ascites due to ovarian cancer were treated with escalating intraperitoneal doses of catumaxomab . the maximum tolerated dose was defined as 10 , 20 , 50 , 200 , and 200 g on days 0 , 3 , 6 , 9 , and 13 . the dose - limiting toxicities were large bowel obstruction common toxicity criteria grade 3 and gamma glutamyl transferase elevation grade 4 . all patients had treatment - emergent adverse events , with fever , nausea , vomiting , abdominal pain , lymphopenia , and general pain being the most common events . in terms of efficacy , 22 of 23 patients did not require any further paracentesis during a follow - up period of up to 37 days . the authors concluded that a dose regimen of 10 , 20 , 50 , and 150 g would be the recommended treatment schedule for further investigation.22 this led to a pivotal phase ii / iii study in patients with symptomatic malignant ascites secondary to epithelial cancers requiring symptomatic therapeutic paracentesis . the study compared paracentesis with intraperitoneal catumaxomab versus paracentesis alone in a two - arm , open - label , randomized trial . the primary endpoint was puncture - free survival , defined as the time to first need for therapeutic puncture or death after treatment . secondary endpoints were time to next paracentesis , ascites signs defined by the patient , ascites signs defined by the investigator , and overall survival . the investigators were to follow an algorithm when a paracentesis was indicated ( ascites > 1l as assessed by a computed tomography scan and signs and symptoms of ascites assessed by the investigator using physical examination and a patient questionnaire ) to ensure a comparable decision on when to perform paracentesis by the different investigators in the different treatment arms . patient in the paracentesis - only treatment group were allowed to cross over to catumaxomab treatment if they still fulfilled the inclusion and exclusion criteria , and had had at least two paracenteses after day 0 of the study . the percentage and outcome of the crossover patients were not reported , although they might influence the secondary endpoint of overall survival . treatment consisted , as recommended , of four constant - rate intraperitoneal infusions at doses of 10 , 20 , 50 , and 150 g of catumaxomab on days 0 , 3 , 7 , and 10 . the antibody was administered via intraperitoneal catheter in an inpatient setting , and the control group was treated with a paracentesis . toxicity was as expected , with predominantly cytokine - release - like symptoms , including pyrexia ( in 60.5% of patients ) , nausea , and vomiting . there were no treatment - related deaths . in total , 258 patients were randomized , of whom 170 received catumaxomab and paracentesis and 88 received paracentesis alone . one hundred and twenty - nine patients had ovarian cancer , and 129 patients suffered from nonovarian cancer , mostly gastric cancer ( n = 66 ) . the primary endpoint of puncture - free survival was significantly prolonged in catumaxomab patients in both strata ( ovarian : 52 versus 11 days , and nonovarian cancer : 37 versus 14 days , p < 0.0001 , respectively ) as well as in the pooled analysis ( 46 versus 11 days ) . the secondary endpoint of median overall survival was not prolonged in the pooled analysis ( 72 days for catumaxomab versus 68 days for paracentesis only , p = 0.0846 ) as well as in the stratified groups . subgroup analysis showed a significant ( p = 0.0313 ) survival benefit for catumaxomab in the gastric cancer patients ( 71 versus 44 days ) . the authors concluded that the treatment regimen demonstrated a clinically relevant benefit in patients with malignant ascites from epithelial cancer.23 catumaxomab , given intraperitoneally in ascending , repetitive doses , prolongs puncture - free survival in patients with malignant ascites . side effects are explained by the mode of action of the drug and are usually reversible . common side effects with intraperitoneal treatment include cytokine release - related symptoms , like fever , chills , nausea , and vomiting .

the progress in new antimicrobial agents to multidrug resistant pathogens for the treatment of various infectious diseases is of increasing interest . therefore different plant extracts of many plants have been used locally against various pathogens , which showed positive results . higher plants and their extracts are used for the treatment of infectious disease as traditional medicines from the very beginning of life . but there are only a small percentage of these plants that have been subjected to pharmacological or biological screening . microbes which are free to move around the world cause different infections in human races . in developing countries about 43 percent of total deaths are due to infectious diseases . who claims significant control in major infectious diseases . due to jumbled use of existing antimicrobial drugs pathogenic bacteria have developed resistance against wide range of antibiotics . the -lactamase producers like staphylococcus aureus , escherichia coli , klebsiella pneumonia , and many others have become a major therapeutic problem , resulting in increased treatment failure and health care cost . microbiologists from all over the world are in search to formulate new antimicrobial drugs and evaluate the efficiency of plant products to replace chemical antimicrobial agents . medicinal plants extracts have shown to serve as a cheap source of antimicrobial agents against pathogenic microbes . these extracts have biologically active compounds , which make them attractive fowler for preying these pathogens . annual postharvest losses are resulting due to insect damage , microbial deterioration , and many other environmental factors such as humidity , aeration , temperature , and cleanliness of the massive storage and are expected to be 1025% of production throughout the world . insects are the major problem in stored grain because they destroy the quantity and quality of the grains . it is claimed that 99 families , 276 genera , and 346 species of medicinal plants have environment- friendly insecticidal properties . plant extracts or pure compounds control insects in several ways , including antifeedant , growth inhibitors , toxicity , mortality suppression of reproductive behavior , and fertility . it is expected that only in america about 200 people are dying every year due to this pesticidal poisoning . these problems resulted in renewed interest in formulating new botanical pesticides , which could be nonhazardous , effective , biodegradable , and of low cost and pose less threat to the environment . the aim of the present study was to evaluate the antibacterial , antifungal , and insecticidal activities of different fractions of oxalis corniculata . these include methanol , n - hexane , chloroform , ethyl acetate , and n - butanol soluble fractions . selection of oxalis corniculata fractions was based on the fact that these fractions were not previously screened for these activities . oxalis corniculata was collected from district buner , kpk , pakistan in june 2012 . nisar ahmad , chairman department of botany , kohat university of science & technology , kohat , kpk , pakistan , and a voucher specimen number 169 was stored there in the herbarium . plant was shade dried at room temperature and 1 kg dried plant materials were soaked in methanol to obtain methanolic extract . extract was evaporated under reduced pressure to dryness ; the residue was weighed ( 81 g ) and redissolved in distilled water . the aqueous solution of the plant extract was subjected to different solvents on the basis of increasing polarity like n - hexane , chloroform , ethyl acetate , and n - butanol to get the respective n - hexane , chloroform , ethyl acetate , and n - butanol solvent soluble fractions with the ratio of 15 g ( n - hexane fraction ) , 22 g ( chloroform fraction ) , 16 g ( ethyl acetate fraction ) , 14 g ( n - butanol fractions ) , and 10 g ( aqueous fraction ) , respectively . all the fractions were dried at low pressure using rotary evaporator and stored at 4c . to evaluate the antimicrobial activity of different fractions of oxalis corniculata , nine microbial species were used , five of which were bacterial pathogens including bacillus subtilis ( atcc7966 ) , staphylococcus aureus ( atcc 12600 ) , escherichia coli ( atcc8677 ) , shigella dysenteriae ( atcc29027 ) , and salmonella typhi ( atcc0650 ) and four fungal species including fusarium solani , aspergillus flexneri , aspergillus flavus , and aspergillus niger were taken from culture collection of department of microbiology , kohat university of science & technology , kohat , pakistan . the bacterial strains were subcultured to get fresh cultures of bacteria . for this purpose a single colony from bacterial strain was inoculated on nutrient broth . 14 g of nutrient agar media was dissolved in 1 l of distilled water at ph 7 and autoclaved for 20 minutes at 121c . the media were allowed to cool down to 45c and poured to petri plates ( 14 cm ) for preparing 75 ml of solid media . using sterile cork borer ( 8 mm ) 7 wells per plate the crude extract and fractions were dissolved in dimethyl sulfoxide ( dmso ) at the same concentration of 2 mg / ml to prepare stock solutions . from the stock solutions , the zones of inhibition of crude extract and fractions were measured in mm after 24 hours of incubation at 37c and compared with the zone of inhibition of standard drug cefixime . four fungal strains including aspergillus flexneri , aspergillus niger , aspergillus flavus , and fusarium solani were tested for the antifungal activity . fungal strains were subcultured in potato dextrose agar ( pda ) and incubated for 7 days at 28c . to evaluate the antifungal activity fungal strains were inoculated on the potato dextrose agar plate ( pda ) by point inoculation . 100 l of solution ( 2 mg / ml in dmso ) , pure dmso ( ve control ) , and antibiotic terbinafin 2 mg / ml ( + ve control ) were used . the insects ephestia cautella and tribolium castaneum were taken from department of zoology , kohat university of science & technology , kohat , kpk , pakistan . 60 mm of petri dish was used to conduct the surface film activity of all the extract by dissolving 50 mg / ml of crud extract and fractions in dmso . extracts were sprayed on to the lower part of the petri dish and allowed to dry out . these treated petri dishes having insects were kept at room temperature in a secured place . the result was observed from time to time starting from 30 minutes to 48 hours and finally recorded . the mortality of insects was confirmed by using simple microscope to check any movement of their organs . in last , the living ( if any ) insects were recovered and submitted to their respective department . the percentage mortality rate was determined by the following formula:(1)mortality% = 100number of survival insamplesnumber of survival in control100 . medicinal plants are the most prominent source of natural products against various common infectious microbes . the appearance of multidrug - resistant infectious microbes , high cost of synthetic compounds , and uninvited side effects of certain drugs insisted the new era to search for the new curative agents from alternative and low cost sources like medicinal plants . the current attempt was made due to increasing resistance of different pathogens ( bacteria and fungi ) and insects against available antibiotics and insecticides , respectively . plants extracts and compounds are of innovative interest as safe and health friendly antimicrobial and insecticidal agents . as a result the different fractions of oxalis corniculata were screened for their antimicrobial and insecticidal activities . to assess the antimicrobial activity of oxalis corniculata , the microbial strains used were tested bacterial pathogens including escherichia coli , bacillus subtilis , staphylococcus aureus , salmonella typhi , and shigella dysenteriae ; the fungal species including fusarium solani , aspergillus flexneri , aspergillus flavus , and aspergillus niger were used . for the evaluation of insecticidal activity the insects used were ephestia cautella and tribolium castaneum . the plant solvent soluble extracts showed a broad spectrum of activities against used microbes and insects . the crude extract , n - butanol , and ethyl acetate soluble fractions showed excellent activities against escherichia coli , salmonella typhi , and bacillus subtilis . similarly crude extract , n - hexane , chloroform , and n - butanol soluble fractions were active against shigella dysenteriae but not active in case of ethyl acetate soluble fraction . similarly the crude extract , n - hexane , and soluble fractions were also found to have significant activity against fungal strains including fusarium solani and aspergillus flexneri ; the crude extract , chloroform , and ethyl acetate soluble fractions were active against aspergillus flavus . the n - butanol soluble fraction was only active against aspergillus flexneri ; also chloroform , ethyl acetate , and n - butanol soluble fractions were inactive against fusarium solani . in case of aspergillus niger investigated the methanol and aqueous extracts of o. corniculata leaves for antibacterial activities against 5 bacterial strains : e. coli , s. aureus , p. aeruginosa , p. vulgaris , and b. subtilis . they recorded zone of inhibition in the range of 614 mm against these pathogens among which b. subtilis showed maximum zone of inhibition ( 14 mm ) , while , in our present investigation , the whole plant ( o. corniculata ) crude extract , its solvent soluble fractions , and the isolated compounds were evaluated for antibacterial and antifungal activities and obtained significant results . the maximum toxicity of plant against ephestia cautella and tribolium castaneum was recorded for the crud extract and chloroform soluble fraction with highest mortality rate especially for the tribolium castaneum ( up to 62% ) . n - butanol soluble fraction also showed valuable mortality ( 46% ) in 48 hours as shown in table 3 . the lowest mortality rate was recorded for ethyl acetate and n - butanol soluble fractions against ephestia cautella ( 08% ) after 48 hours ; however it was nonactive till 24 hours . the comparative study shows that the plant is more toxic against tribolium castaneum than ephestia cautella . there is no literature available on the insecticidal activity of oxalis species to compare with the present results . the chloroform soluble fraction was subjected to column chromatography and as a result two known compounds , compound 1 ( figure 1 ) and compound 2 ( figure 2 ) , were isolated whose structures were elucidated with the help of ei , hreims , 1d , and 2d nmr techniques . the two isolated compounds 1 and 2 were subjected to antibacterial , antifungal , and insecticidal activities . the result showed that compound 2 was more active than compound 1 against the tested organisms . both the compounds were active against all the tested bacteria except s. aureus as shown in table 4 . while compounds 1 and 2 showed negative activity against fusarium solani , aspergillus flavus , aspergillus niger , and aspergillus flexneri which indicates that these compounds are inactive against these species of fungi . table 5 shows that insecticidal activity of compound 2 was also potent as compared to that of compound 1 . the present study suggests that oxalis corniculata has good antibacterial , antifungal , and insecticidal properties and can be used for the treatment of infections and control of insects . the plant extracts could be a new source for antibiotics and pesticides with minimum noxious effects on the environment . further studies may also lead to isolate and characterize the active compounds of the plant extracts and to elucidate their biological mechanisms of action .

ceruloplasmin ( cp ) a positive app is increased due to stimulation of macrophages and monocytes at the sites of inflammation by cytokines . pleural fluid ( pf ) accumulates in exudative effusions due to alteration of local factors influencing the formation and absorption of pf ; in contrast , transudative effusion is due to alteration of systemic factors . studies have shown that cp is significantly increased in exudative pe compared to other apps . so the aim of the study was to know the diagnostic value of pfcp and serum ceruloplasmin ratio ( cpr ) in differentiating pe into exudate and transudate as compared to light 's criteria . patients were recruited from the medical wards , who were undergoing thoracocentesis for the definitive diagnosis of pe between february and july 2011 . based on previous studies sensitivity of the light 's criteria was taken as 98% and sensitivity for cp in pf in one of the studies by calikoglu et al . expecting at least 85% sensitivity for cp in our setup and with 80% power and alpha error of 0.05 , we need to study 71 cases . seventy - one consecutive patients with pleural effusion ( pe ) who gave informed consent and fulfilled the inclusion and exclusion criteria were included in the study . all the patients admitted with pe for diagnostic thoracocentesispatients who gave the informed consent . all the patients admitted with pe for diagnostic thoracocentesis patients who gave the informed consent . patients on drugs like anticonvulsants ( carbamazepine , phenobarbital , phenytoin , valproic acid ) , oral contraceptive pillspregnant womenpatients with previously diagnosed wilson 's disease . patients on drugs like anticonvulsants ( carbamazepine , phenobarbital , phenytoin , valproic acid ) , the definitive diagnosis made by the clinicians was obtained from the patient medical records after prior approval . para pneumonic effusion was considered when there was acute febrile illness with purulent sputum , pulmonary infiltrates , responsiveness to antibiotic treatment , or identification of the organism in the pf by culture . tuberculous pleurisy was diagnosed with a positive acid fast stain in pf , pleural biopsy or sputum ; presence of caseous granulomas in pleural biopsy or clearance of effusion in response to antitubercular therapy . pe due to pancreatitis was diagnosed based on the definitive diagnosis of pancreatitis and the absence of other causes of pe . pleural effusion due to congestive heart failure was determined by an enlarged heart , pulmonary venous congestion on radiograph , peripheral edema , response to chf treatment , and the absence of malignancy or pulmonary infiltrates associated with an inflammatory process or any other cause of pe . renal failure was diagnosed when there was raised serum urea and creatinine values , signs of fluid overload and absence of any other causes of effusion . the pf was collected by thoracocentesis for the estimation of total protein ( tp ) , lactate dehydrogenase ( ldh ) and cp . venous blood sample was collected from the patient into a vacutainer bd ( becton dickinson ) , was allowed to clot for 20 to 30 min and centrifuged at 3000 rpm for 10 minutes . pleural fluid and serum was used to measure tp , ldh and stored at -20c degrees for measurement of cp . tp was measured in both serum and pf using the modified biuret method using bichromatic end - point technique . both the analytes were measured in dade dimension rxl max - siemens health care diagnostics limited . the cp was measured manually using copper oxidase method and the absorbance was measured at 530 nm using a spectrophotometer and calculation was done to give the results in mg / dl . after the measurement of tp and ldh , pf was divided into exudate and transudate by light 's criteria ( tp ratio > 0.5 , ldh ratio > 0.6 , ldh greater than 2/3 of the upper limit of normal for serum ldh ) . roc curve was used to determine the cut - off value with best sensitivity and specificity for the pfcp and cpr , and divide the pf into exudates and transudates . the number of patients diagnosed as exudate and transudate by the light 's criteria , pfcp and cpr was compared with the definitive diagnosis . results on continuous measurements are presented as mean 1sd , median and quartile ( 25 , 75 ) , and results on categorical measurements are presented in number and percentage ( % ) . the mann - whitney u test has been used to find the significance of study parameters on continuous scale between the two group 's exudates and transudates . a cut - off value to differentiate the pf into exudate and transudates depending on the highest sensitivity and specificity for the pfcp and cpr was determined by the roc curve . all the patients admitted with pe for diagnostic thoracocentesispatients who gave the informed consent . all the patients admitted with pe for diagnostic thoracocentesis patients who gave the informed consent . patients on drugs like anticonvulsants ( carbamazepine , phenobarbital , phenytoin , valproic acid ) , oral contraceptive pillspregnant womenpatients with previously diagnosed wilson 's disease . patients on drugs like anticonvulsants ( carbamazepine , phenobarbital , phenytoin , valproic acid ) , oral contraceptive pills patients with previously diagnosed wilson 's disease . the definitive diagnosis made by the clinicians para pneumonic effusion was considered when there was acute febrile illness with purulent sputum , pulmonary infiltrates , responsiveness to antibiotic treatment , or identification of the organism in the pf by culture . tuberculous pleurisy was diagnosed with a positive acid fast stain in pf , pleural biopsy or sputum ; presence of caseous granulomas in pleural biopsy or clearance of effusion in response to antitubercular therapy . pe due to pancreatitis was diagnosed based on the definitive diagnosis of pancreatitis and the absence of other causes of pe . pleural effusion due to congestive heart failure was determined by an enlarged heart , pulmonary venous congestion on radiograph , peripheral edema , response to chf treatment , and the absence of malignancy or pulmonary infiltrates associated with an inflammatory process or any other cause of pe . renal failure was diagnosed when there was raised serum urea and creatinine values , signs of fluid overload and absence of any other causes of effusion . the pf was collected by thoracocentesis for the estimation of total protein ( tp ) , lactate dehydrogenase ( ldh ) and cp . venous blood sample was collected from the patient into a vacutainer bd ( becton dickinson ) , was allowed to clot for 20 to 30 min and centrifuged at 3000 rpm for 10 minutes . pleural fluid and serum was used to measure tp , ldh and stored at -20c degrees for measurement of cp . tp was measured in both serum and pf using the modified biuret method using bichromatic end - point technique . both the analytes were measured in dade dimension rxl max - siemens health care diagnostics limited . the cp was measured manually using copper oxidase method and the absorbance was measured at 530 nm using a spectrophotometer and calculation was done to give the results in mg / dl . after the measurement of tp and ldh , pf was divided into exudate and transudate by light 's criteria ( tp ratio > 0.5 , ldh ratio > 0.6 , ldh greater than 2/3 of the upper limit of normal for serum ldh ) . roc curve was used to determine the cut - off value with best sensitivity and specificity for the pfcp and cpr , and divide the pf into exudates and transudates . the number of patients diagnosed as exudate and transudate by the light 's criteria , pfcp and cpr was compared with the definitive diagnosis . results on continuous measurements are presented as mean 1sd , median and quartile ( 25 , 75 ) , and results on categorical measurements are presented in number and percentage ( % ) . the mann - whitney u test has been used to find the significance of study parameters on continuous scale between the two group 's exudates and transudates . a cut - off value to differentiate the pf into exudate and transudates depending on the highest sensitivity and specificity for the pfcp and cpr was determined by the roc curve . a total of 71 patients were included in the study of which 7 patients were excluded as a definitive diagnosis was not reached . patient 's pf were classified into exudate ( 58 patients ) and transudate ( 6 patients ) by definitive diagnosis , which was obtained from the patient health records . the mean age of the population was found to be 50.96 yrs and the mean value of the tp , cp and the median values of the ldh in serum and pf were 6.22 g / dl , 46.28 mg / dl , 285 iu / l and 3.55 g / dl , 24.31 mg / dl , 326.5 iu / l , respectively . the mean age of exudates and transudates was 50.15 yrs ( range 18 - 82 years ) and 58.83 yrs with age range 34 - 69 years , respectively . different causes of pleural effusion in exudates and transudates with their frequency and percentage shown in table 2 are the mean values of tp , cp and median values of ldh in both exudates and transudates in serum and pf . there was a significant difference between the two groups in the mean values of pftp , pfcp , pfldh and cpr by the mann - whitney u test as shown in table 3 . compared to definitive diagnosis the sensitivity of light 's criteria was 94% and 83% , respectively . mean and sd value of total protein , ldh , ceruloplasmin in serum and pleural fluid between two groups significance of test parameters between exudate and transudate figure 1 is the roc curve for the determination of pfcp cut - off values and figure 2 is the roc curve for the cpr . the area under the curve for pfcp was 0.914 and with a cut - off value of 13.34 mg / dl showed highest sensitivity and specificity to differentiate the pf into exudate and transudate . similarly , the area under the curve for cpr was 0.94 and with cut off 0.33 having highest sensitivity and specificity to differentiate the two groups . shown in tables 4 and 5 is the sensitivity and specificity of the pfcp and cpr at various cut - off values . sensitivity , specificity , ppv and npv of the light 's criteria , pfcp and cpr is shown in table 6 . roc = receiver operator characteristics curve , pfcp = pleural fluid ceruloplasmin , pf = pleural fluid . the area under the curve for the pleural fluid ceruloplasmin is 0.914 roc curve of the cpr for the detection of exudates in pf . roc = receiver operator characteristics curve , cpr = pleural fluid to serum ceruloplasmin ratio , pf = pleural fluid , the area under the curve for the cpr is 0.94 sensitivity and 1-specifity at various cut off value for pleural fluid ceruloplasmin as obtained by roc curve sensitivity and 1-specifity at various cut - off values for pleural fluid to serum ceruloplasmin ratio as obtained by roc curve sensitivity , specificity , ppv , npv of light 's criteria , pfcp and cpr the sensitivity and specificity of pfcp was 89% and 83% which was lower compared to light 's criteria . similarly , the sensitivity and specificity of cpr were 91% and 83% which were better than pfcp alone . the first step in the evaluation of a pe is to differentiate into exudate and transudate . light 's criteria misidentify a transudative pe as exudative pe in as many as 25% of the cases and the criteria include measurement of tp , ldh in both serum and pf to differentiate the two groups . several other parameters like pf cholesterol greater than 44.85 mg / dl showed a sensitivity and specificity of 97.1 and 100% , respectively . studied on apps and found that sensitivity and specificity of cp ( 92% and 84% ) and transferrin ( 84% and 80% ) was better than other apps . g / l is useful in differentiation of exudates and transudates when the patient is on diuretic therapy . pleural fluid to serum bilirubin concentration ratio greater than 0.6 showed sensitivity and specificity of 96% and 83% , respectively . have concluded that light 's criteria is superior to pf bilirubin , cholesterol and albumin gradient in differentiation of exudate and transudates . cp is a copper - containing protein with ferroxidase activity , which is responsible for the oxidation of fe ( ferrous iron ) into fe ( ferric iron ) , therefore assisting in its transport in the plasma in association with transferrin , which can only carry iron in the ferric state . it is also a positive app , where the protein levels increase in response to inflammatory cytokines . fleming et al . studied rat lung and found that cp mrna is expressed in rat lung on exposure to endotoxin and the specific site of production of cp is alveolar macrophages . they also found expression of cp mrna in the lung on hyperoxic induction in rats . have shown that the cp is secreted by human peripheral blood monocytes on specific induction by inf-. this explains the increased level of cp in exudative pf as seen in our study , where the cause is inflammatory injury of the pleura . in our study studies done by doustjalali et al . in 2006 have patients with nasopharyngeal carcinoma showing enhanced serum and tissue cp expression and pousset et al . in 2001 showed high levels of cp in the serum of transgenic mice developing hepatocellular carcinoma . pe in acute pancreatitis is caused mainly due to transdiaphragmatic lymphatic blockage or pancreaticopleural fistulae and an increased concentration of il-1 , il-6 and tnf- in pancreatic secretion . studied 80 patients with pe on apps like c - reactive protein , haptoglobin , transferrin , alpha-1acid glycoprotein and cp . cp concentrations were determined by immunoturbidometrical methods ( cobas integra 700 , roche diagnostics , mannheim , germany ) . they found the pfcp in exudates was 27 10 mg / dl similar to our study values ( 25.91 13.04 mg / dl ) . in transudates they found a higher value ( 16 2 mg / dl ) compared to our study ( 8.86 5.24 ) . this may be due to lower percentage of the people with transudate and with different population group in our study . they had 26 transudative effusions with ccf . compared to light 's criteria , pfcp has less sensitivity and npv but had similar specificity in differentiating exudative pe from transudative pe . transudate pe are not related to local pleural pathology , but are produced by an imbalance between the hydrostatic and oncotic pressures , which does not lead to inflammation . in this study we found that pfcp and cpr were able to differentiate exudates from transudates . the light 's criteria showed similar positive predictive value ( 98.2% ) and better negative predictive value ( 62.5% ) compared to pfcp ( 45% ) and cpr ( 50% ) respectively in differentiating exudates from transudates . it was also found that the pleural fluid to serum ceruloplasmin ratio was better than pfcp alone . holmberg - laurell factor was used in the estimation of ceruloplasmin by ferroxidase method and crystalline cp was not used in standardization . in inflammation there may be increase in total ceruloplasmin , whereas ferroxidase method estimates only holo ceruloplasmin . pleural fluid from patients with no diagnosis was excluded from the study ; these are the cases where there is a need for extensive investigations and a need for an effective marker to classify pf into exudate or transudate . a larger study with more transudates

oral mucosa is an active site for the onset of numerous opportunistic infections such as oral candidiasis , epstein barr virus ( ebv ) associated oral hairy leukoplakia , kaposi sarcoma , periodontitis , and other ulcerative lesions in hiv infected patients [ 18 ] . the loss of mucosal immunity in the oral mucosa is generally thought to contribute to this process . cd4 t cells play a key role in oral mucosal immunity and numerous studies have documented the loss of cd4 t cells in the gastrointestinal tract ( git ) such as the small and large intestinal mucosa during hiv and siv infections [ 914 ] . the advent of highly active art ( haart ) has led to a decrease in the incidence of oral infections in some groups of patients suggesting that haart likely restores some of the mucosal immunity that is lost during hiv infection . it is not clear if the perturbations of cd4 t cells seen in the lower git during hiv infection occur to a similar extent in the oral mucosa and if haart effectively restores cd4 t cells in the oral mucosa . cd4 t lymphocytes play an important role in the generation and maintenance of both humoral and cellular immune responses by providing t cell help . unlike peripheral tissues , the cd4 t cells in the git display a predominantly memory phenotype ; these cells are critical for the generation of secondary immune responses to the previously exposed pathogens in these tissues . though numerous studies have evaluated peripheral cd4 t cell counts and associated them with onset of opportunistic infections , few studies have examined the dynamics of cd4 t cells in oral mucosa during hiv infection and haart . cd4 t cells in the oral mucosa , as in other mucosal tissues , are probably highly susceptible to hiv infection and their loss likely compromises the integrity of oral immune system . we sought to characterize the nature and phenotype of cd4 t cells in the oral mucosa and determine if cd4 t cell dynamics were altered during hiv infection and antiretroviral therapy ( art ) using the siv infected nonhuman primate model . rhesus macaques infected with siv has been a valuable model to study hiv pathogenesis , and studies have shown siv infected macaques display similar oral pathologies and susceptibilities to opportunistic infections such as ebv and candidiasis as seen in hiv infected subjects [ 1517 ] . our results show that the prevalence and phenotype of cd4 t cells in the oral mucosa mirror those of cd4 t cells in the rectal mucosa , with a repopulation of cd4 t cells during art . interestingly , a fraction of cd4 t cells repopulating the oral mucosa were found to harbor siv dna suggesting that the infected viral reservoir continues to persist in oral mucosa during therapy . archived samples from rhesus macaques ( macaca mulatta ) of indian origin were used for this study . the animals were housed in accordance with the american association for accreditation of laboratory animal guidelines and were seronegative for siv , simian retrovirus ( srv ) , and simian t cell leukemia virus ( stlv ) type 1 . peripheral blood and oral and rectal mucosal samples were obtained from five healthy animals , eight animals chronically infected with siv , and four animals that were treated with antiretroviral drugs pmpa ( tenofovir ) and ftc ( emtricitabine ) continuously for more than 6 months until sacrifice . pbmc was isolated by density gradient centrifugation and mucosal samples were processed by enzymatic digestion followed by enrichment over a percoll gradient as described previously [ 10 , 18 ] . plasma viral loads were determined by real - time pcr with an abi prism 7700 sequence detection system ( applied biosystems ) using reverse - transcribed viral rna as template using methods previously described . isolated cells were labeled with a combination of cd3-cy7apc , cd4-pb , cd28-cy5-pe , cd95-fitc , and cd8-alexafluor700 and analyzed by flow cytometry using procedures described previously [ 10 , 12 , 18 , 2022 ] . all the antibodies were obtained from bd biosciences ( san diego , ca ) and titrated using rhesus macaque pbmc . labeled cells were fixed with 0.5% paraformaldehyde and analyzed using a becton dickinson lsr ii flow cytometer . for analysis of siv - gag dna in cd4 t cells , cells were labeled as above and cd4 t cells were sorted using a bd facs aria sorter . il-17 production in cd4 t cells was determined after short - term stimulation with 10 ng / ml of pma ( phorbol myristate acetate ; sigma - aldrich , saint louis , mo ) and 500 ng / ml of ionomycin ( sigma - aldrich , saint louis , mo ) in the presence of brefeldin - a ( bd biosciences ) for 4 hours . after stimulation , cells were labeled with anti - cd3-cy7apc , cd8-alexa-700 , and cd4-pe . cells were fixed in cytofix / perm buffer ( bd biosciences ) and labeled with anti - il-17-apc ( e - biosciences ) . labeled cells were fixed in 0.5% paraformaldehyde and analyzed using a becton dickinson lsr ii flow cytometer . siv - gag dna in cd4 t cells was determined using a highly quantitative pcr assay for siv - gag as previously described using siv - gag specific primers and probes . the assay was calibrated using a cell line that carried only a single copy of proviral siv dna . processed samples were analyzed using an abi 7500 taqman pcr instrument ( applied biosystems inc . ) . flow cytometric data were analyzed using flowjo version 9.2 ( tree star , inc . , statistical analysis was performed using t - test with graphpad prism version 4.0 software ( graphpad prism software , inc . , san diego , ca ) . we first examined the prevalence of cd4 t cells in the oral mucosa of healthy animals to determine if they differed from that of other tissues such as the rectal mucosa and peripheral blood ( figure 1(a ) ) . peripheral blood was found to have a higher proportion of cd4 t cells as compared to cd8 t cells ; ratio of cd4 t cells to cd8 t cells was ~2 : 1 ( figure 1(b ) ) . in contrast , cd4 t cells constituted a minor population of t cells in the oral mucosa with a majority of t cells being cd8 t cells . as such the ratio of cd4 t cells to cd8 t cells was significantly inverted ( < 0.5 ) as compared to peripheral blood and resembled that of the rectal mucosa . previous studies have shown that mucosal cd4 t cells displayed a predominantly memory t cell phenotype [ 10 , 12 , 22 , 24 , 25 ] . to determine if phenotype of cd4 t cells in the oral mucosa differed from those of the rectal mucosa and periphery , we examined the expression patterns of cd28 and cd95 on cd4 t cells from the oral mucosa of healthy rhesus macaques and compared them to cd4 t cells in the rectal mucosa and peripheral blood ( figures 2(a)-2(b ) ) . nave and memory cd4 t cells were delineated based on the differential expression of cd28 and cd95 with memory cd4 t cells expressing high levels of cd95 as compared to little or no expression of cd95 on nave cd4 t cells [ 10 , 12 , 21 , 22 , 26 ] . our results showed that nave cd4 t cells were the predominant cd4 t cell subset in peripheral blood as has been previously reported . in contrast to peripheral blood , however , cd4 t cells in the oral mucosa expressed a predominantly central memory t cell phenotype with most cd4 t cells coexpressing cd28 and cd95 . the phenotype of cd4 t cells in the oral mucosa resembled that of cd4 t cells found in the rectal mucosa . we next examined the prevalence of cd4 t cells in the oral mucosa of rhesus macaques that were chronically infected with siv and compared them to rectal mucosa and peripheral blood cd4 t cells . siv infected animals had ~6 logs of plasma viremia ( figure 3(a ) ) , and chronic siv infection was associated with a significant depletion of cd4 t cells in both the oral and rectal mucosa ( figure 3(b ) ) . as peripheral blood harbors a heterogeneous mix of nave and memory cd4 t cells , and memory cd4 t cells like their counterparts in the oral and rectal mucosa are the primary targets for infection , we examined the dynamics of central memory cd4 t cells in peripheral blood during chronic siv infection and compared them to oral and rectal mucosal cd4 t cells . we have previously shown that memory cd4 t cells in peripheral blood and lymph nodes were significantly depleted to a similar extent as cd4 t cells in the jejunum of siv infected animals . in line with these studies , chronic siv infection was associated with a significant loss of memory cd4 t cells in peripheral blood . to examine if art was accompanied by a repopulation of cd4 t cells in the oral mucosa , we determined the prevalence of cd4 t cells in the oral mucosa during art and compared them to the rectal mucosa and peripheral blood . art was associated with a significant suppression of plasma viral loads to levels that were below the limit detection ( figure 3(a ) ) . cd4 t cells were found to significantly repopulate the oral and rectal mucosa though the level of repopulation in the rectal mucosa was significantly lower than levels seen in healthy animals ( figure 3(b ) ) . peripheral blood central memory cd4 t cells were found to significantly rebound after art . as t - helper-17 ( th17 ) cells play an important role in oral mucosal immunity [ 20 , 27 ] , we examined the expression of il-17 in cd4 t cells repopulating the oral mucosa during art using intracellular staining and flow cytometry . our results showed that art was associated with restoration of th17 cells with ~40% of cd4 t cells in the oral mucosa expressing il-17 suggesting that art was capable of restoring oral mucosal immunity . to determine if cd4 t cells repopulating the oral mucosa harbored infected cells , we examined the level of siv dna in sorted cd4 t cells from the oral mucosa of siv infected animals during art ( figure 3(d ) ) . cd4 t cells repopulating the oral mucosa carried ~4000 copies of siv dna/10 cd4 t cells suggesting that low levels of the siv infected latent viral reservoir persisted in the oral mucosa during therapy even after complete suppression of plasma viremia . we were unable to sort cd4 t cells from infected untreated animals due to the near total depletion of cd4 t cells during chronic siv infection . the oral mucosal environment is home to numerous opportunistic pathogens that remain under tight homeostatic control of the immune system . this control breaks down during hiv infection leading to the onset of various opportunistic infections and inflammatory conditions . cd4 t cells play a central role in maintaining mucosal immune homeostasis and their loss in the gastrointestinal mucosa has been extensively documented . surprisingly , however , limited studies have examined in detail either the loss or repopulation of cd4 t cells during infection and therapy or characterized the nature of cd4 t cells resident in the oral mucosa relative to other mucosal tissues . our studies provide evidence that oral mucosa resembles other mucosal tissues such as the rectal mucosa as compared to peripheral blood ; both oral and rectal mucosa had an inverted cd4 : cd8 ratio as compared to peripheral blood ( figure 1(b ) ) . interestingly , however , the oral mucosa was found to have significantly fewer cd4 t cells than the rectal mucosa . like the cd4 t cells in the rectal mucosa , cd4 t cells in the oral mucosa of healthy animals had a predominantly central memory phenotype with few nave cd4 t cells ( figures 2(a)-2(b ) ) suggesting that akin to the rectal mucosa the oral mucosa likely serves as an effector lymphoid compartment . siv infection was accompanied by a near total loss of cd4 t cells in the oral mucosa that mirrored their loss in the rectal mucosa suggesting that oral mucosal immunity is significantly compromised as in the git during chronic stages of infection . numerous studies have documented the loss of cd4 t cells in mucosal tissues during hiv and siv infections [ 9 , 10 , 1214 , 24 , 25 ] . continuous art was accompanied by a repopulation of cd4 t cells in both the oral and rectal mucosa though the extent of repopulation in the rectal mucosa was significantly lower than the proportions of cd4 t cells prevalent in the rectal mucosa of healthy animals . as we did not sample tissues longitudinally , it is difficult to determine if sustained repopulation of cd4 t cells occurs in the oral mucosa during art . numerous longitudinal follow - up studies of hiv infected subjects using long - term highly active art ( haart ) have , however , shown that cd4 t cells fail to continuously repopulate the mucosa even after 10 years of continuous haart even though transient repopulation is apparent during therapy [ 2832 ] . on the other hand , guadalupe et al . showed that there was a substantial delay in cd4 t cell repopulation of the gut mucosal tissues during haart . longitudinal sampling of oral mucosal tissues during art would be essential to address this question . interestingly , repopulating cd4 t cells in the oral mucosa were found to harbor ~40% of th17 cells suggesting that art was capable of restoring oral mucosal immunity . showed that effective repopulation of the gut lymphoid tissue was characterized by enhanced th17 responses , whereas klatt and brenchley suggested that effective repopulation of th17 cells in the gastrointestinal mucosa was associated with better disease prognosis . th17 cells play a key role in maintaining mucosal immune homeostasis and immunity and repopulation of these cells in the oral mucosa can potentially lead to better oral health during art . several studies [ 20 , 3538 ] have shown that the loss of th17 cells was associated with translocation of microbial products and increased immune activation suggesting that repopulating th17 cells may potentially limit translocation across oral mucosal surfaces . interestingly , ~2% of repopulating cd4 t cells in the oral mucosa were found to be infected and carry viral dna ; there were ~4,000 copies of siv dna/10 cd4 t cells . previous studies have shown that low levels of viral replication continued in the mucosal tissues of hiv infected patients and siv infected macaques during highly suppressive art [ 30 , 31 , 3941 ] . these findings suggest that , like other mucosal tissues , the infected viral reservoir persists in the oral mucosa during art . our findings have significant implications for development of functional cure therapies that target the eradication of the oral hiv reservoir in patients undergoing highly suppressive haart . in conclusion , our studies provide novel insights into the dynamics of cd4 t cell depletion and repopulation in the oral mucosa during chronic siv infection and therapy . cd4 t cells repopulating the oral mucosa harbor th17 cells suggesting that art can potentially restore immunity at oral mucosal surfaces . the persistence of low levels of infected viral reservoir , however , suggests that art regimens likely need to be combined with other approaches to eradicate the oral viral reservoir leading to better and more effective immune reconstitution in the oral mucosa during hiv infection .

odontogenic tumors comprise of a complex group of lesions of diverse histopathological types and clinical behavior . of all swellings of the oral cavity , 9% are odontogenic tumors and within this group , ameloblastoma accounts for 1% of lesions . who defines it as a locally invasive polymorphic neoplasia that often has a follicular or plexiform pattern in a fibrous stroma . its behavior has been described as being benign but locally aggressive . in 20% of all cases the tumor can be found in the upper jaw , predominantly in the canine or molar region . within the mandible , 70% are located in the molar region or the ascending ramus , 20% in the premolar region and 10% in the anterior part . the age range is usually between the first and the seventh decade of life with a mean in the fourth decade . clinically , ameloblastomas can be classified into 4 groups : unicystic , solid or multicystic , peripheral , and malignant . the unicystic ameloblastoma usually appears as a cystic lesion with either an intraluminal or an intramural proliferation of the cystic lining . multicystic ameloblastoma can infiltrate into the adjacent tissue and has the ability to recur and even metastasize . it is a soft - tissue version of an ameloblastoma but can also involve the underlying bone . it is defined as an ameloblastoma that has already metastasized but still maintains its classical microscopic features . a histological classification subdivides into follicular , plexiform , acanthomatous and granular ameloblastoma . in most cases the tumor is asymptomatic , presenting as an incidental finding on orthopantomography . the most common symptoms are facial swelling , pain , malocclusion , loosening of teeth , ill - fitting dentures , periodontal diseases or ulceration , oroantral fistulas and nasal airway obstruction . literature basically describes two therapy strategies : a conservative way of treatment and radical procedures . while smaller lesions are generally treated by a less aggressive approach , larger lesions require a radical surgical tumor ablation resulting in large defects making reconstruction difficult . management of ameloblastoma has been controversial because of the unique biological behavior of this disease as a slow - growing , locally invasive tumor with a high rate of recurrence.[1217 ] recurrence rates of ameloblastoma are reportedly as high as 15 - 25% after radical treatment[1315 ] and 75 - 90% after conservative treatment.[1317 ] therefore , wide resection of the jaw in accordance with the treatment of malignant tumors is usually recommended for ameloblastomas . recent advancements in understanding the biological behaviors of ameloblastoma have led to more rational surgical approaches.[1820 ] in total , 20 patients with primary ameloblastoma treated during the period from 2003 to 2008 were available for this study . clinical information and radiographs were obtained from the records of the department of oral and maxillofacial surgery , institute of dental sciences , bareilly . the patient age ranged from 15 to 60 years , with a sex distribution of 12 males and eight females . according to the clinical and radiographical features , the distribution of patients of each type was seven unicystic , five multicystic and eight solid types . two cases occurred in the maxilla and 18 in the mandible and the tumors were often located in the molar to the ramus region [ table 1 ] . histological diagnosis and classification were based on the criteria defined by the world health organization ( who ) histological classification . the methods of treatment consisted of radical surgery ( segmental resection ) and conservative treatments ( enucleation with bone curettage ) . radical surgery was defined as the procedure in which the ameloblastoma was resected , with a safety margin of at least 2 cm of normal bone , with or without a continuity defect . conservative treatment has been carried out in accordance with our comprehensive conservative treatment protocol for ameloblastomas since the 1980s [ table 2 ] . small lesions are submitted to excisional biopsy , and once the ameloblastoma has been diagnosed , the lesion is enucleated and curetted , including the surrounding healthy bone . lesions of solid - type tumor with clear margin viewed by means of radiographical examination are usually curetted extensively , and lesions with unclear margins , such as those with a soap - bubble appearance , or those with ineffective marsupialization are subjected to marginal or segmental resection depending on their size and location . comprehensive treatment protocol of ameloblastoma enucleation with bone curettage was defined as the procedure in which the ameloblastoma was enucleated in conjunction with excision of the overlying mucosa and , subsequently , sufficient bone curettage . while curetting the bone , 2.5% gentian violet was used on the surface of adjacent healthy bone to ensure removal of the tumor . the colored bone was usually curetted three or four times , for more than 5 mm in depth , by using a large round bur . if an isolated tumor nest was recognized in the cancellous bone during this procedure , additional curettage was performed . when the nerve was exposed in the surgical field , it was lifted out from the bony canal when curetting the bone to avoid damage to the nerve . after the surgical treatment , the patients had clinical and radiographical examinations every year for at least 5 years . the effects of resection on the recurrence data after a follow - up period of at least 5 years were evaluated . furthermore , these recurrence data were analyzed with respect to clinical types and who patterns . out of the 20 ameloblastomas , 18 were located in the mandible ( 90% ) and two in the maxillary region ( 10% ) . twelve of the mandibular ameloblastomas ( 66.6% ) were found in the corpus- and mandibular - angle region , both of them reaching the ascending ramus . in six cases ( 33.3% ) the size of the lesions varied between 4 and 8 cm in diameter , as is seen in figures 1 and 2 . ( a ) preoperative frontal view , ( b ) multilocular radiolucency involving right body and angle of mandible , ( c ) resected part of mandible involving 2 cm of normal bone , ( d ) reconstruction of mandible with iliac crest graft , ( e ) postoperative x - ray , ( f ) postoperative frontal view ( a ) ameloblastoma of right body of mandible causing expansion , ( b ) opg showing multilocular lesion at right body of mandible , ( c ) resected part of mandible involving 2 cm of normal bone , ( d ) postoperative x - ray ten patients underwent enucleation with bone curettage , including those with six unicystic and four multicystic types of ameloblastoma . the effect of the marsupialization was graded radiographically as follows : extremely effective , the lesion almost disappeared ; effective , the lesion decreased to less than half its initial size ; ineffective , the lesion did not decrease remarkably or tended to grow further . the effect of enucleation with bone curettage was evaluated as extremely effective in one case , effective in six cases and ineffective in three cases . extremely effective cases were observed only in the unicystic type , whereas effective and ineffective cases were included in both the unicystic and the multicystic types . the effective rate , including extremely effective and effective cases , was 70% ( 07/10 ) in total [ table 3 ] . effect of enucleation and curettage for cystic ameloblastoma types of surgical treatment and recurrence data for primary ameloblastoma are shown in table 4 . recurrences were observed in seven patients ; these included one case with segmental resection and six cases with enucleation and bone curettage . in terms of comparison of recurrence rates of different surgical modalities , relatively high recurrence rates were observed in the patients treated by enucleation with bone curettage ( 60% ) . types of surgical modalities for primary ameloblastoma wound healing disturbances occurred in only one patient after mandibular reconstruction . wide resection of the jaw is usually the recommended treatment for ameloblastoma , should priority be given to the recurrence rate . however , radical surgery often means that the patients have serious complications including facial deformity , masticatory dysfunction , and abnormal jaw movement . considering the characteristics of ameloblastoma as a locally invasive but slow - growing and extremely rare metastasizing benign tumor , the priority of the treatment method should be discussed from the points of morbidity and quality of life of the patients , noting that the recurrence rate is not always the primary factor . two therapy strategies are mentioned in literature : a conservative way of treatment and radical procedures . non - radical surgical procedures like enucleation and curettage , combined with liquid nitrogen spray cryosurgery , or just drilling of the perilesional bone are mentioned to be useful in unicystic ameloblastomas , especially in children and young patients . other authors show high rates of reccurence of ameloblastoma after conservative treatment protocols and therefore recommend radical surgical treatment . authors suggests a rational radical conservative resection of the mandible with preservation of the lower border of the mandible to maintain the continuity of the lower jaw and the facial contours . in the previous reports , conservative treatments for ameloblastoma appeared to have failed to control local recurrences . sehdev et al , reported recurrence after the conservative approach ( curettage ) in more than 90% of 92 ameloblastomas . shatkin and hoffmeister reported that 86% of 20 mandibular ameloblastomas recurred after curettage compared with a 14% recurrence rate after en bloc resection . other authors have reported a series of 84 ameloblastomas in which they found a 52% rate of recurrence in patients treated conservatively and a 25% rate of recurrence in patients with primary tumor treated by the radical approach . however , extensive tumors require a more radical approach . the amount of resection is variable and depends on the site and extension of the tumor . patients included in our study all presented with locally advanced tumors already infiltrating the surrounding soft tissue . according to our opinion , conservative ways of treatment in these cases are not appropriate and will surely result in local recurrent tumors making further surgical treatment even more complicated resulting in cosmetic and speech deficits . when planning the treatment of ameloblastoma , it is important to understand the growth characteristics and to remove the full extent of the tumor , including the surrounding tissues . otherwise , the remaining tumor cells may lead to multiple morbidities of recurrence . recent advancements in the understanding of the biological behaviors of ameloblastoma have revealed that unicystic lesions are well - localized by the fibrous capsule of the cyst , with few tumors broaching peripheral tissues , whereas multicystic and solid lesions are characterized by an aggressive infiltration to adjacent tissue . he stated that the recommended treatment for solid and multicystic ameloblastoma was radical treatment , whereas unicystic ameloblastoma was usually cured by curettage . guideline for evaluation of growth characteristics of tumors postsurgical defects in the maxillary region predispose the patient to hypernasal speech , fluid leakage into the nasal cavity , impaired masticatory function , and in some patients , various degrees of cosmetic deformity . mandibular resection can also prove devastating to mastication , deglutition , phonation , and oral competence . moreover , the mandible frames the lower third of the face and represents a major component of the human appearance . satisfactory reconstruction of complex jaw defects , especially in a single - step procedure , is therefore a surgical challenge . for benign tumors , the bone grafts have become a reliable source during the last few years in osseous reconstruction . the fibula , scapula and iliac crest are the commonly chosen donor sites to reconstruct mandibular or maxillary defects . for reconstruction of defects in the mandible we preferred iliac crest bone grafts as a good quality of bone furthermore , the natural curvature of the iliac bone is ideally suited for mandibular reconstruction , using the ipsilateral or contralateral crest , depending on which segment of the mandible needs rebuilding . the donor defect should be carefully closed , since a potential for abdominal herniation still remains . some authors recommend using nonvascularized grafts for reconstruction of mandibular continuity defects less than 9 cm in length . according to the authors this technique allows better results concerning facial esthetics and implant insertion . the success rate , however , was low and patients receiving nonvascularized grafts had to undergo an average of one more surgical procedure for total reconstruction . according to our opinion free flaps with well vascularized soft tissue provide reliable wound healing , reducing the risk of plate exposure . it is a well - known fact that nonvascularized bone transplants show high resorption rates resulting in severe bone loss after a few months because of lack of physiological stress . in our case the quality and height of bone after removal of the osteosynthesis material was fairly well . in our follow - up regime , patients were scheduled for clinical and radiological examination twice a year for the first 5 years and after that only once a year . we suggest a long follow - up period for at least 10 years as recurrence may also appear years after primary surgery . ameloblastoma has a high rate of local recurrence if it is not adequately removed . in our opinion , radical surgical resection of ameloblastoma is the treatment of choice . especially in cases of large , expansive tumors a radical surgical protocol is a very good option to prevent relapse of the tumor on a long - term basis . reconstruction of the defects with bone graft material allows good functional and esthetic outcome and decreases the number of surgeries . for reconstructing the mandible we prefer bone grafts from the iliac crest . the natural curvature and variable bone height offers the possibility of exact reconstruction of the defect .

it is well - known that the molecular modeling of many biological processes requires a rigorous treatment of the long - ranged electrostatic interactions . furthermore , recent years have seen the development of many promising next - generation force fields and fast ab initio methods that endeavor to generalize the electrostatic interactions to higher - order atomic multipoles . specific examples include the following : polarizable force fields , such as amoeba ; density - based force fields , such as gem and s / g-1 ; force fields that model the perturbative or many - body expansion of the energy ; hybrid quantum mechanical / molecular mechanical ( qm / mm ) methods ; and molecular orbital - based quantum mechanical force fields ( qmffs ) , such as the embedded fragment model , x - pol , and the closely related modified the use of higher - order atomic multipoles in these examples , and many other models , offer the promise of improved accuracy , but at a larger computational cost . in order to elucidate the strengths and weaknesses of proposed models , it is necessary to make comparison to experiment , which often requires their application within molecular simulations under periodic boundary conditions . at the same time , these applications provide empirical measures of the preliminary model s true computational cost . therefore , fast electrostatic algorithms must be developed that are equipped to handle generalized charge densities , so that new models can be applied and tested . the ewald , particle mesh ewald ( pme ) , and fast fourier poisson ( ffp ) methods are three algorithms used to evaluate the long - ranged electrostatic interactions of periodic systems . these methods were originally designed to accommodate systems consisting of point charges ; therefore , modifications to their original formulations are required to apply them to models employing multipolar charge densities . in the present work , we derive a unified set of equations for the ewald , pme , and ffp methods using a spherical tensor gradient operator formalism that extend these methods to arbitrary multipole order . expressions for the energies , forces , and the generalized multipolar potentials required to incorporate the electrostatics into the qmff self - consistent field ( scf ) procedure are provided . previous works have extended these methods within the framework of cartesian point multipoles and cartesian gaussians , as opposed to the solid harmonic multipoles considered here . other related works were not formulated to arbitrary multipole order or led to a formulism that did not prove to be computationally efficient . the efficiency of our formulation is demonstrated through qmff molecular dynamics applications described below and extended in part 2 of this series . during preparation of this manuscript , simmonett has independently reported an extension of pme for use with solid harmonic multipoles that shares many characteristics with the pme method presented here ; however , our analysis extends significantly beyond ref ( 52 ) by comparing the accuracy and performance of ewald , pme , and ffp methods as stand - alone electrostatic methods and upon implementation within a linear - scaling qmff . section 2 describes the generalized charge density ; derives equations for the generalized ewald , pme , and ffp methods ; and discusses net charge corrections , error analysis , and the integration of the methods into the mdc qmff . section 3 compares the accuracy and computational cost of the methods , as a function of multipolar order and within the context of condensed - phase molecular simulations of water . our goal is to compute the electrostatic interaction of a neutral charge density (r ) with itself and its periodic images:1where the unit cell defining the periodicity is described by the lattice vectors a1 , a2 , a3 and reciprocal - space vectors a1 * , a2 * , a3 * ; and the periodic images are replicated by integer lattice translations n = n1a1 + n2a2 + n3a3 . the energy ( given by eq 1 ) is finite only when the charge density is neutral ; however , a widely used approximation for the treatment of charged systems will be discussed in section 2.6 . furthermore , we adopt the standard convention of removing the infinite self - energy of point charges ( and point multipoles ) whenever those energies may appear . upon replicating all integer translations of the aperiodic density , (r ) , the edges of a lattice with the same shape , orientation , and dimensions can be redrawn about any origin ; and the periodic density filling each cell will contain exactly one instance of (r ) that appears to have been wrapped into the cell s interior . the apparent wrapping of (r ) to the cell boundary is an illusion formed by the effluence of density emanating from the other translations , upon making (r ) periodic . the aperiodic density is not required to be confined within a primary unit cell to achieve this effect . nor is it a requirement for the evaluation of eq 1 , because the electrostatic potential of n(r + n ) is periodic . the methods presented in this work are solutions to eq 1 for a density composed of atom - centered point multipole expansions . the two most common ways being cartesian multipoles or those based on spherical harmonics . our definition of a multipole moment is the inner product of a density with a real - valued regular solid harmonic:2an auxiliary basis of charge density l(r ) , satisfying3is thus well - suited to describe an atomic multipole expansion . the definition of cl(r ) is closely related to the scaled regular solid harmonics frequently encountered in fast multipole methods . in brief , the real - valued scaled regular solid harmonics rlmc / s(r ) are the real rlmc(r ) = rerlm(r ) and imaginary rlms(r ) = imrlm(r ) components of the complex - valued scaled regular solid harmonics:4where5is an associated legendre polynomial . because the complex - valued harmonics are symmetry related rlm*(r ) = ( 1)rl , m(r ) , the real - valued harmonics are fully described by the set of non - negative m values . the notation is simplified , when appropriate , through the introduction of a greek subscript whose sign merely acts to symbolize the cosine / sine designation6the real - valued regular solid harmonics7differ only by factors8that are chosen to reproduce racah s normalization9 in the interest of writing the expression for a point multipole , we begin by deducing the form of a gaussian multipole function from eqs 3 and 9:10where11is a gaussian monopole , a = { d / dxa , d / dya , d / dza } are gradients with respect to the gaussian center , and cl( ) is a spherical tensor gradient operator ( stgo ) . a stgo is constructed by replacing the cartesian coordinate arguments of the solid harmonic with their corresponding cartesian derivative operators . the present work specifically makes use of the stgo chain - rule identity,12which follows from the homogeneity of solid harmonics cl(ar ) the application of eq 12 to the last line of eq 10 establishes the relationship between l(r ra ) and a gaussian monopole . a dirac delta function is a gaussian monopole in the limit of infinite exponent:13and a point multipole is this limit applied to eq 10:14thus , a sum of atom - centered point multipole expansions is 15 methods for solving eq 1 avoid the infinite sum of explicit lattice translations by approximating a periodic density with a finite number of plane wave basis functions r|k er plane waves can be used as a basis to represent real , periodic functions:18where ck = ak + ibk . complex numbers have been introduced to compactly express two independent linear least - square fit problems : the representation of the even ( cosine ) and odd ( sine ) character of the density . one can show that the two least - square fit solutions for ak and bk are equivalent to having chosen19to minimize the sum of squared errors | , defined by20 in other words , eq 19 is identical to having determined ak and bk by solving the two ( uncoupled ) equationsandrespectively . by integrating over the volume of a unit cell , the first line of eq 19 effectively integrates one instance of (r ) that has been wrapped into the cell s interior . the second line of eq 19 exploits the periodicity of the plane waves by unwrapping the density and modifying the integration limits accordingly . one can deduce that cos(x ) = cos(x ) and sin(x ) = sin(x ) imply the symmetries a k = bk , and c k = ck*. as a consequence of these symmetries , and , therefore , the sum over allk in eq 18 naturally acts to cancel the imaginary components from each term . that is , although each term is complex , the other half of the sum adds its conjugate . the appearance of re in our description of ewald , pme , and ffp is to remind the reader that the imaginary numbers vanish and only the real component of each term thus needs to be computed . equation 18 is often referred to as a complex - to - real reverse ( or inverse ) fourier transform and is typically implemented within computer software to utilize only a subset of symmetry - unique ck * values that are provided as input . weighted least - squares fits can be performed to generalize the plane wave expansion described above . these fits produce coefficients21that minimize the sum of squared errors || weighted by a linear hermitian operator . in the general case , the components of ck store the result of a fit that may have required a coupled ( as opposed to independent ) solution to ak and bk . for the specific case r||r = |r r| , eq 21 is called an electrostatic fit . the coefficients of an electrostatic fit reduce to eq 19 , because the plane waves are eigenfunctions of the coulomb operator ( eq 16 ) . in other words , eq 19 are the plane wave expansion coefficients that best reproduce the electrostatic potential of the periodic density . one could , in principle , directly project (r ) into the periodic basis and compute the interaction from the plane wave representation ; in practice , however , this is intractable because an infinite number of plane waves would be required to model (r ) . therefore , the ewald method abandons the direct solution of eq 1 , preferring instead to decompose the periodic density into smooth and discontinuous components:22whereby the smooth model density composed of gaussians23is well - approximated from a linear least - squares fit to reasonably few plane waves:24furthermore , the model density was chosen to reproduce the long - range electrostatic potential of the point multipoles to ensure that only short - ranged corrections are required . in our notation , k| and k| = e/4 ) the ewald energy is the interaction of (r ) with the electrostatic potential of the plane wave projected periodic gaussian density (r ) ( see eq 32 ) upon correcting the short - ranged differences between the point and gaussian potentials (r ) ( see eq 27):26where pa , l = |/qa , l and pa , l = |/qa , l are the corresponding multipolar potentials . the real - space corrections to the potential , energy , and multipolar potential are given as follows : potential correction : 27energy correction : 28multipolar potential correction : 29where30these corrections are assumed to be sufficiently short - ranged , such that only nearby minimum image separations rab and |r rb| need to be considered . an efficient algorithm for computing eq 30 and its gradients , particularly for low - order harmonics , is described in ref ( 56 ) . the application of that algorithm requires the following definition of the auxiliary vector:31where fn(x ) is the boys function . the reciprocal - space ewald potential , energy , multipolar potential , and contribution to the gradient are readily obtained upon noting |k = 1.reciprocal-space ewald potential : 32energy : 33multipolar potential : 34contribution to the gradient : 35the extension of ewald to atom - centered point multipole expansions presented here differs from the standard point - charge ewald method only by the inclusion of stgos in the structure factor and real - space correction . a linear - scaling implementation of the real - space ewald corrections requires only short - range cutoffs and a sufficiently large gaussian exponent ; however , by fixing the cutoff and , hence , the exponent the computational complexity of the reciprocal - space ewald potential then scales with o(n ) , because(1 ) the number of plane waves must increase with the size of the system , to maintain consistent resolution of the periodic density , and(2 ) the calculation of each sk requires a summation over the number of particles.in the unlikely event that the particles happened to be positioned such that they formed a uniformly spaced grid , however , then the expression for sk would become a sum over grid points ( see eq 48 ) and the contribution of all grid points to all sk could be computed o(n log n ) using a fast fourier transform ( fft ) . the novelty of pme is its coercion of the particle positions to exploit this otherwise unlikely scenario to speed the calculation of the reciprocal - space potential and energy . the number of plane waves must increase with the size of the system , to maintain consistent resolution of the periodic density , and the calculation of each sk requires a summation over the number of particles . pme approximates the particle positions with a linear combination of predefined uniformly spaced fft grid points rt,36that can be used to compute fourier coefficients from numerical quadrature . the gaussian quadrature of plane waves is equivalent to simpson s integration rule performed on a regular grid:37where fk is a discrete fourier transform ( dft):38 t is the index of the grid ( t = ( t1 , t2 , t3 ) ) and rt is the location of a fft grid point : n1 , n2 , and n3 are the number of fft grid points in each lattice direction ; n = n1n2n3 . is a cardinal b - spline weight evaluated about a particle position:39 in our notation , the argument of mn(u ) is meant to be periodically wrapped into the range ( nd/2,nd/2 ] . mn is a cardinal b - spline function that holds recursion properties for efficient evaluation of their values and gradients.40the b - spline weights are real r| = |r , have even symmetry r| = r| , sum to one ( trt r| = 1 r ) , and are nonzero only for those grid points near the particle . the number of grid points contributing to the approximate position of a particle is determined from the chosen b - spline order without regard to the fft grid spacing . following the work of schoenberg , previous descriptions of pme have used cardinal b - splines as a linear basis to construct exponential euler splines . in that point of view , the structure factors are computed from approximate plane waves that are spline - evaluated at the actual particle positions . we below choose an alternate perspective by applying eq 36 to obtain the same outcome , which we then relate to ref ( 38 ) . the charge density is composed of atom - centered functions ; therefore , it is convenient to re - express eq 36 as41which is deduced from the invariance of the relative particle and grid positions upon their simultaneous reflection about the origin and subsequent translation by + r ; that is , ra r ra and rt r rt . an atom - centered gaussian is thus approximated by a linear combination of gaussians centered about the fft grid points:42however , this has the unintended consequence of making the approximate gaussian appear artificially diffuse as the b - spline order is increased . fortunately , if the b - spline is of sufficient order to be accurately integrated by the discrete fourier transform , then the fourier coefficients of the approximate gaussian43are those of the true gaussian,44scaled by the b - spline dft coefficients k . note that k = k * , because the b - splines are even functions . repeating this procedure for , or any function |f , yields analogous results . in other words , the fourier coefficients of an approximately positioned function should be scaled by k . let us continue on a brief aside that relates eq 36 to the exponential euler splines used in ref ( 38 ) by considering its application to k|k = k , kv in the manner above using either of the relations45the result vk|rr|k dr = kk|k implies the need to effectively renormalize an approximate plane wave upon grid interpolation ; that is , the relationship between eq 36 and the exponential euler splines is the interpretation of whether it is the approximate plane wave or the fourier coefficient of the approximately positioned function that is renormalized:46the subtle distinction between these interpretations has no practical consequence on the pme method other than , perhaps , how the mathematical terms are grouped together ; eq 47 scales the structure factor by k , whereas ref ( 38 ) absorbs it into the definition of sk . the periodic representation of the model gaussian density ( eq 23 ) composed of the approximate gaussians ( eq 42 ) is , upon scaling the fourier coefficients,47where the pme structure factor,48is the forward dft of49whose evaluation avoids o(n ) operations by the locality of (r ) . the pme reciprocal - space potential is the electrostatic potential of eq 47,50and yields the following pme reciprocal - space energy , multipolar potential , and contribution to the gradient : pme reciprocal - space energy : 51multipolar potential : 52contribution to the gradient : 53the extension of pme to atom - centered point multipole expansions presented here differs from the standard point charge pme method only by the inclusion of stgos acting upon the b - spline in eqs 4951 and , like ewald , the real - space correction ( eqs 27 and 28 ) . although the stgo has many remarkable properties when acting upon a spherical function , a regular or irregular solid harmonic , or a product of the these functions , the b - spline weights can not take advantage of these properties . instead , one must treat the stgo as a linear combination of gradient operators,54whose coefficients are exactly those that relate clmc / s(r ) to cartesian monomials:55where56and o = 0 and o = 1 . in the above notation , m = || , cl0( ) poisson ( ffp ) , like ewald , abandons the direct solution to eq 1 by introducing a gaussian density whose potential can be determined from a plane wave expansion . unlike the ewald method , ffp evaluates the plane wave expansion coefficients from numerical quadrature to take advantage of ffts . the electrostatic potential of the plane - wave - resolved gaussian density is computed in fourier space , evaluated on a regular grid by means of a reverse fft , and then used to numerically integrate the coulomb self - energy of the gaussian density . therefore , the ffp real - space corrections account for the short - ranged differences between the gaussian and point interaction energies:57the plane wave representation of the periodic model density composed of the gaussian multipoles is58where59the evaluation of rt| avoids o(n ) operations by evaluating l(rt ra ) only for minimum image distances within a suitable cutoff . the real - space energy corrections , tl,j , vanish when the gaussians do not overlap60where sn,0a , b = 1 n10n20n30ab ) . therefore , eq 60 is well - approximated by computing the integrals for the short - ranged , minimum - image rab separations only . explicit expressions for the real - space energy correction , multipolar potentials , and gradient contribution are analogous to those shown in eqs 2831 upon substituting with /2 , which arises from the gaussian product theorem . the ffp reciprocal - space energy , multipolar potential , and contribution to the gradient are as follows : ffp reciprocal - space energy : 61multipolar potential : 62contribution to the gradient : 63the gradient of the gaussian basis,64is efficiently computed using the following derivative properties:656667when negative m values are encountered , the reader is implicitly instructed to apply the symmetry property rl , mc / s(r ) = (1)rlmc / s(r ) , which follows directly from rlm*(r ) = ( 1)rl , mc / s(r ) , where the plus / minus sign ( ) corresponds to the cosine / sine designation . let r||r = n|r r + n| ; then , we can rewrite eq 1.68if the net charge is nonzero ( (r ) dr = q ) , then the system is nonphysical and this definition of the energy is infinite . although it is possible to neutralize the density in arbitrarily different ways to produce different energies , a particularly convenient choice is made by introducing a uniform background density,69and n(r + n ) = q / v , everywhere . with this choice of neutralization , the energy is70however , /2 = /2| , as written in the previous sections , by virtue of excluding the k = 0 term in the ewald summation , because it is charge - neutral . similarly , /2 vanishes , because the exclusion of the k = 0 term produces a potential composed of functions that are each orthogonal to a constant over the range of the unit cell . the last term in eq 70 vanishes , by symmetry , for all nonzero multipoles and is well - approximated by an integral over all space if is sufficiently large to extinguish the correction potential within the bounds of the unit cell ( in a minimum image context ) . as a result of these properties , the energy of a charged system is71an analogous result is written for ffp by replacing with /2 . for completeness , we note that it has been pointed out that eq 1 is absolutely convergent only if the total dipole moment of the system is zero ; however , it is otherwise only conditionally convergent . in other words , the asymptotic value of eq 1 is dependent on the shape of the supercells used to replicate the system . term 2||/[(2s + 1)v ] can be applied to mimic an infinite spherical sample of a cubic cell ; however , this energy is usually ignored in ewald implementations , because its application to charged systems has a dependence on an arbitrary origin and it produces discontinuous energy changes when charged residues are wrapped into the primary unit cell . by ignoring this term , we are said to employ tinfoil boundary conditions , because it is equivalent to embedding the crystal inside a perfect conductor ( s ) . figure 1 compares the ewald energy ( eq 26 ) to a brute force evaluation of eq 1 , as a function of replicated unit cell length . the fundamental unit cell is a 10 cube containing three - point multipoles:72where r = ( 1 ) . the unit cell is replicated by extending the sides of the cell to form increasingly larger cubes . the relative energy error is defined aswhere e is the ewald energy evaluated with |kmax| = 256 and en is eq 1 after replication to the specified size . the relative error is bounded by 10 , because of our use of double - precision arithmetic . the loss of one or two additional digits of precision should be expected , because of the accumulation of round - off errors . comparison between the ewald energy to the energy computed from explicit replication of a unit cell , as a function of replicated cell length for systems composed of the indicated multipole orders . figure 24 make use of an ad hoc three - site water model to analyze the relative force errors and the real - space and reciprocal - space timings of ewald , pme , and ffp , as a function of atomic multipole order . these three figures are the analysis of a box of 1024 waters whose bulk density has been equilibrated with tip4p - ew at 298 k. the 3-site multipolar water models used to perform the analysis are systematically constructed from a 5-site charge - only water model . the 3- and 5-site water models use the dftb3 gas - phase structure of water ( these coordinates are superimposed onto the tip4p - ew waters ) : roh = 0.957143 , rhh = 1.572691 , hoh = 110.4812 ( the 5-site model includes two additional charges located at the o the atom charges ( a.u . ) ( qo5-site = 0.12404869708 , qh5-site = 0.66106645662 ) and the bond charges ( qb5-site = 0.59904210808 ) were computed from the isolated water dftb3 density matrix ; that is , qo5-site is the one - center atomic orbital ( ao ) product contribution to the mulliken charge of o , and qb5-site is the mulliken bond charge between o and h. the multipole moments of the 3-site model are constructed from a mulliken - like partitioning and subsequent solid harmonic translation of the 5-site model bond charges:7374the intrawater interactions are excluded when analyzing the relative force errors with these models . lmax indexes the ad hoc 3-site water model being considered , as distinguished by the multipole expansion order on the oxygen . wall clock time required to evaluate the real - space corrections ( eq 29 ) for a box of 1024 waters using different real - space cutoffs . the inset values are timing ratios t(lmax)/t(0 ) , that is , the slow - down relative to the charge - only model . reciprocal - space evaluation timings ( left column ) , relative force errors ( rfes ) ( middle column ) , and the gaussian exponents ( right column ) chosen to minimize the rfes of ewald ( top row ) , pme ( middle row ) , and ffp ( bottom row ) , using the box of 1024 waters . lmax indexes the ad hoc 3-site water model being used . in order to interpret the results in figure 4 , it is useful to understand how the multipole order influences the magnitude of the forces . for this purpose , figure 2 decomposes the intermolecular electrostatic forces within the 1024 water box by the percent contributed from the charge charge interactions , the non-chargecharge interactions , and those interactions that involve a particular angular momentum l. let fl be the 3 n vector of atomic forces for a system of waters composed of the ad hoc 3-site water model using an order l multipole expansion . the percent contribution of the charges , the multipoles , and a particular multipole l to the forces are respectively chosen to be757677 the relative force errors ( rfes ) in figure 4 are calculated using the expression78where fref , l is the vector of ewald forces evaluated with |kmax| = 128 and whose real - space corrections include all n minimum image interactions . the gaussian exponent has been nonlinearly optimized to minimize each rfe , and their values are shown in figure 4 using the convention = . the timings reported in figures 3 , 4 , and 6 were performed on an intel xeon e5520 2.27 ghz workstation consisting of a total of 8 cores , and the software was rudimentarily parallelized with openmp to make use of all cores . the analyses in figures 5 , 6 , and 7 , and the data shown in table 1 , were produced from condensed - phase calculations using the mdc linear scaling quantum force field . in brief , the quantum mechanical treatment of the entire system is replaced by a series of quantum calculations for each molecule . although the molecular orbitals of each fragment are not directly coupled , the subsystems remain coupled through the interactions of their electron densities and empirical potentials . specifically , the mdc method performs dftb3/3ob semiempirical calculations of each molecule while subjecting them to an effective chemical potential arising from the intermolecular interactions . this idea is conceptually similar to density functional embedding theory ; however , mdc replaces the rigorous evaluation of the embedding potential with computational tractable empirical approximations that are tuned for high accuracy . the standard dftb3 semiempirical model evaluates the electrostatic interactions from mulliken charges that are updated until self - consistency is reached . in the mdc formalism described below , the standard dftb3 treatment continues to be used for those atoms within a common fragment ; however , we concoct an auxiliary set of atomic multipoles from the density matrix to improve the intermolecular interactions . the multipolar potentials arising from those interactions enter the fragment fock matrices as an effective external chemical potential , and the multipoles are updated at each step of the scf procedure . let ea be the dftb3 energy for molecule a consisting of atoms located at ra ; then , the mdc energy is79where na is the number of electrons in fragment a , qa , l is an atomic multipole moment determined from the dftb3 density matrix , and pa , l = einter/qa , l is the multipolar potential describing the effective external potential experienced by atom a. with this convention , the -spin fock matrix used to construct the orbitals of molecule a is given as80as explained in ref ( 31 ) , the atomic charges are computed from a biased mulliken partition of the density matrix , and the higher - order multipole moments ( up to quadrupoles for heavy elements ) are computed from the single - center ao product components of the density matrix . the intermolecular interaction energy einter consists of lennard - jones potentials and the electrostatic interaction of the atomic multipoles . the electrostatic energy and multipolar potentials are evaluated using either the ewald , pme , or ffp methods described in the previous sections . because the multipole moments are dependent on the density matrix and the fock matrix is dependent on the interactions with the other molecules , the mdc energy must be optimized until a mutual convergence is met for all molecules . conservation of the mdc total energy for 1 million steps in a nve simulation of n , n - dimethylglycine , using the multipolar pme method with 6th order b - splines . scf and scf + gradient timings using the mdc quantum force field for a series of water boxes . l = 0 indicates that only atomic charges were used to compute electrostatics , whereas l = 2 indicates that up to quadrupoles were used on the heavy atoms . rfes observed with the mdc linear - scaling quantum force field using various methods of computing the electrostatic interactions . the top portion of the figure shows a box of 1024 waters , whereas the bottom of the figure shows a supercell of n , n - dimethylglycine containing 4608 atoms . errors are reported relative to ffp evaluated with 3 points / and a minimum image ( min img . indicates the range used for the short - range real - space corrections and , for ffp , the evaluation of the model gaussian density at the fft grid points . dotted lines indicate the ability of the electrostatics protocol to reproduce the electrostatic component of the force , using the atomic multipoles obtained from the reference ffp calculation . the dashed lines are mdc rfes , using cutoff - based electrostatics without any ewald treatment . sixth - order b - spline interpolation , grid spacing of 1 point / , and short - range cutoff of 9 . figure 5 illustrates the quality of the mdc forces by demonstrating conservation of the total energy in a nve simulation of an n , n - dimethylglycine ( dmg ) crystal at 225 k. the crystal consists of 288 dmg residues ( 4608 atoms ) constructed from supercell replication of the experimentally determined x - ray structure and was simulated at the experimental density with a locally modified development version of pmemd that incorporates the mdc method . the simulation was performed using a 0.5 fs time step for 1 million steps , and the electrostatics were computed with sixth - order b - spline pme , a 9 real - space cutoff , and a 1 pt / fft grid density . figure 6 demonstrates linear scaling of the mdc method for a series of water boxes . in addition , timings were recorded for an ad hoc mdc model that limits the intermolecular interactions to monopoles for the sole purpose of determining the relative cost of incorporating atomic quadrupoles . the pme calculations were performed with sixth - order b - splines , a real - space cutoff of 9 , and an fft grid density of 1 point / . the ffp calculations were performed with an fft grid density of 1 point / and a cutoff of 9 in both the real- and reciprocal - space calculations , that is , the evaluation of the model gaussian density at the fft grid points . unlike the ad hoc water models used in figure 4 , mdc self - consistently models each molecule s polarization in response to its environment ( the other molecules ) . because the polarization is induced from the environment s electrostatic potential , the electrostatic protocol indirectly introduces atomic force errors by altering the electronic polarization . within the mdc method , the change in polarization is produced from the difference in the multipolar potential contributions to the fock matrix . figure 7 decomposes the mdc force errors to quantify the extent to which the protocols indirectly effect the forces through their difference in polarizations . first , a mdc calculation is performed using a reference electrostatic protocol to produce a scf converged energy ( emdc , ref ) and charge density ( qref ) . the dotted lines in figure 7 are the rfes computed from the vectors of intermolecular electrostatic forces evaluated with qref8182where eelec , model and eelec , ref are the model and reference intermolecular electrostatic energies , respectively . a second mdc calculation is then performed using a model electrostatic protocol throughout the scf procedure to produce a converged energy emdc , model and charge density qmodel . the solid lines in figure 7 are the rfes computed from8384which are evaluated with different electrostatic protocols and charge densities . the dashed lines in figure 7 are computed from eqs 83 and 84 , where emdc , model is evaluated using a molecule - based cutoff electrostatic protocol ; that is , no treatment for periodicity was applied beyond employing the minimum image convention within the cutoff radius . the electrostatic interactions between the waters were smoothly switched off from 8 to 9 , based on the o o separation . all reference calculations were performed with ffp using a 3 point / grid density and a full minimum image treatment of the short - range corrections and reciprocal - space evaluation . the pme and electrostatic cutoff methods are compared in table 1 , which examines how these methods effect the density and heat of vaporization of liquid water at 298 k. these properties were computed from 8-ns simulations of 512 waters in the npt ensemble using the monte carlo barostat , and the andersen thermostat , as implemented in pmemd . a time step of 1 fs was used in conjunction with shake to constrain the internal structure of the waters to the isolated dftb3 geometry . the parameters of the mdc water model used to generate these properties are described in part 2 of this series . figure 1 establishes the correctness of the ewald formulas for various multipole orders by comparing its energy to brute force evaluation of eq 1 . analogous plots for ffp and pme with suitably dense fft grids and b - spline order are indistinguishable from the ewald results shown in figure 1 . the errors in the energy flatten to a constant in the range of 1010 , because the large number of calculations involved in the brute force evaluation suffer from a loss of numerical precision . as the multipole order is increased , the energy converges more quickly , because the coulomb interactions decay by r. when l 4 , the range of the interactions become so small that the energy is well - approximated by the minimum - image interactions . before comparing the ewald , pme , and ffp rfes , it is useful to briefly discuss the relative magnitude of the forces associated with increasing the multipole order . as shown in figure 2 , charge interactions represent 40%50% of the force when higher - order multipoles are used in the ad hoc water model . furthermore , higher - order multipoles progressively contribute less to the force , since their interactions become shorter - ranged . all interactions involving the octupoles within the lmax = 3 water model , for example , contribute < 5% to the force , whereas all interactions involving l = 5 contribute < 0.5% . the wall - clock time required to evaluate the real- and reciprocal - space contributions to the multipolar potentials , as a function of multipole expansion order , are displayed in figures 3 and 4 , respectively . the real - space corrections evaluated from eqs 29 and 30 and the algorithm in ref ( 56 ) are particularly efficient for lmax 3 . for higher - order expansions , techniques based on rotations into an internal - coordinate system can be used to further improve performance ; however , considering the short - range and relative insignificance of these interactions , it can be verily questioned if practical models for condensed - phase simulations will consider their additional cost worthwhile . including quadrupoles on the oxygen causes the real - space evaluation timings to slow down by a factor of 1.8 , relative to a charge - only model . comparison of the reciprocal - space timings in figure 4 suggests that , for this system of 1024 waters , ffp and ewald are 2 and 100 times slower than pme , respectively , for comparable error levels . furthermore , the evaluation of the real - space corrections at lmax = 2 with a cutoff of 9 are approximately twice as slow as the sixth - order b - spline pme reciprocal - space evaluation times . the optimized gaussian exponents shown in figure 4 are far more sensitive to the choice of real - space cutoff than they are to multipole order . as the real - space cutoff is increased , the gaussian exponent decreases to become better resolved in the plane wave basis , and this consequently decreases the errors of the reciprocal - space forces . similarly , increasing the pme b - spline order affords the opportunity to increase the gaussian exponent to reduce the errors in the real - space correction without sacrificing accuracy in the reciprocal - space potential . much of this manuscript uses the word error to describe a difference between an accurate electrostatics protocol and a more approximate treatment . however , this phrasing should not be misconstrued to incorrectly suggest that the pme gradients are inconsistent with the pme energy , for example . to emphasize this , figure 5 demonstrates conservation of total energy in a nve simulation of dmg for 1 million time steps evaluated with the mdc quantum force field , whose if there were an inconsistency between the energy and forces , then we would observe a significant drift in the energy as time is propagated ; however , we do not observe a drift . the mdc method is linear scaling when using either pme or ffp , as illustrated in figure 6 . when evaluated with pme , a box of 4096 waters can be scf converged within < 0.7 s. furthermore , our timings indicate that the use of quadrupoles slows the mdc calculation by a factor of 1.5 , relative to a charge - only model . this is slightly less than the ratio observed in figure 3 , because the mdc and charge - only mdc evaluations share common operations that are independent of the multipole order used in their intermolecular electrostatics ( for example , their fock matrix diagonalizations ) . the number of scf cycles required to reach convergence is not particularly sensitive to the size of the system , which can be inferred from the linear - scaling shown in figure 6 . the scf convergence rate is largely determined by the quality of the initial guess orbitals . in practice , the difference between md steps is so small that convergence can typically be reached within 46 cycles . ewald , pme , and ffp are approximate , but their accuracy can be systematically improved by adjusting the number of plane waves , the gaussian exponent , and the real - space correction cutoff . in the analysis of figure 4 , we examine how these parameters alter the electrostatic energy protocol by quantifying their effect on atomic forces ( for a fixed charge density ) , e/r|q , while ignoring their effect on electrostatic potentials e/q|r . however , the parameters do effect the electrostatic potentials . therefore , the parameters indirectly effect the charge density through the propagation of electrostatic potential errors within the scf , which cause its convergence to a different density matrix . in other words , not only will ab initio methods ( or any polarizable model ) suffer from the primary error associated with the partial derivative e/r|q , they also incur a secondary error when the model and reference forces are evaluated about different charge densities . the dotted lines in figure 7 are the primary electrostatic errors ; that is , they are the rfes associated with the electrostatic forces evaluated from the atomic multipoles generated from the reference calculation . the solid lines in figure 7 contain both the primary and secondary errors produced when the reference and approximate electrostatic protocols are used throughout the scf procedures to yield two slightly different densities . the reader may first notice that the primary ffp errors decrease and converge , with respect to fft grid density quickly ; however , the secondary errors trail off at 10 to 10 . the errors in figure 7 were generated with a 10 tolerance on the maximum value appearing in the error matrix , that is , the commutator between the fock matrix and the density matrix . as expected , one observes a decrease in error as the pme b - spline order is increased , the fft grid density is increased , and as the real - space cutoff is increased . the mdc rfes observed in water and dmg parallel the primary errors ; however , the dmg secondary errors are significantly larger than those observed in water . the reason for this is because a dmg molecule contains 16 atoms , whereas water contains 3 . by having more aos , the dmg molecules are more likely to converge to a slightly different density matrix . before pme was popularized in md simulations , electrostatic cutoffs were often used , and many of the original water models were thus parametrized using a cutoff protocol . it was later found that application of those water models with pme caused significant changes in the thermodynamic properties of liquid water , and it has been suggested that new water models not be parametrized using cutoffs , because their application within simulations of biomolecules often necessitates the use of an ewald treatment . nonetheless , some recently developed water models have continued to be parametrized using cutoffs . therefore , we are obliged to investigate by how much this may effect condensed - phase properties . first , in figure 7 , we note that a cutoff of 9 produces an rfe value of 0.1 , in comparison to the rfe value observed with a sixth - order b - spline pme with a grid spacing of 1 point / fft ( 2 10 ) . furthermore , even if all n minimum image interactions were computed without pme , the rfe is still 0.04 . finally , we performed md simulations using a parametrized mdc water model to obtain an equilibrated density and heat of vaporization , as shown in table 1 , and we reperformed the simulations using switched - cutoff electrostatics to make comparison . in agreement with previous works , we observe that cut - off electrostatics cause the density of water to increase , and because the interactions in this range are attractive overall , this also causes the heat of vaporization to increase . this work presented extensions of the ewald , particle mesh ewald ( pme ) , and fast fourier poisson ( ffp ) methodologies to systems composed of point multipole expansions to arbitrary order by making use of the spherical tensor gradient operator . the timings and errors inherent to these methods were compared using ad hoc water models and with a parametrized water based on the modified divide - and - conquer ( mdc ) linear - scaling quantum force field . these comparisons lead us to conclude that ( i ) the ffp method is approximately twice as slow as the pme method at comparable error levels ; ( ii ) the inclusion of quadrupoles in the linear - scaling force field slow the calculations by 1.5 , relative to a charge - only model ; and ( iii ) with the exception of the ewald method , the real - space corrections are more expensive than the reciprocal - space calculations for typical cutoff values . furthermore , our results suggest that the evaluation of multipolar electrostatics involving orders greater than 3 could likely be computed to an acceptable error using an electrostatic cutoff , because of their overall short - range and relative insignificance , in comparison to lower - order interactions . the relative force errors exhibited within mdc were decomposed into primary and secondary errors , where the primary errors directly result from the approximations within the pme or ffp algorithms for a given density , and the secondary errors are the propagation of the model s electrostatic potential within the self - consistent field ( scf ) procedure , resulting in a different converged density matrix . it is found that the force errors closely follow the primary errors , and the magnitude of the secondary errors is related to the number of atomic orbitals ( aos ) . nevertheless , the presence of these errors does not imply that the mdc forces are inconsistent with its energy , which was demonstrated with an nve simulation that was devoid of an energy drift . instead , these differences merely reflect how similar an electrostatic protocol is to another reference protocol . finally , the importance of using an ewald treatment in simulations , as opposed to using electrostatic cutoffs , was emphasized by comparing the density and heat of vaporization of water . the electrostatic cut - off method was found to artificially increase the density and heat of vaporization of water .

chronic obstructive pulmonary disease ( copd ) is characterized by persistent airflow limitation and is most often degenerative in nature.1 for this reason , prevention of disease progression is an important therapeutic target in copd patients . although forced expiratory volume in 1 second ( fev1 ) shows only weak correlation with the clinical course of copd , fev1 has been accepted as a surrogate marker for disease progression in many clinical trials.2 unfortunately , no single pharmacologic treatment has been shown to conclusively reduce the annual rate of fev1 decline . in the uplift trial , in which the primary outcome measured was the rate of mean fev1 decline , both before and after bronchodilation ( bd ) , in copd patients with an fev1 of < 70% of the predicted value , tiotropium did not significantly reduce the annual rate of fev1 decline.3 however , the drug did seem to reduce the rate of post - bd fev1 decline in a post hoc analysis of a patient subgroup with an fev1 of between 50% and 70% of the predicted value.4,5 this suggested that the ability of tiotropium to prevent copd progression differs between stages . furthermore , the result may be related to the different rates of fev1 decline that occur at various stages of copd : the rate has been reported to be faster in early - stage of copd than at a later stage . bridevaux et al6 reported that the mean annual decline in fev1 was 40 ml / yr for patients with global initiative for chronic obstructive lung disease ( gold ) stage 1 copd , whereas it was 20 ml / yr for those with stage 24 copd . this suggests that the effect of tiotropium on the deceleration of fev1 decline may be more noticeable in patients with gold stage 1 copd , and that it may be masked in advanced copd patients with a slower rate of fev1 decline . however , only a few studies have included patients with an fev1 of > 70% of the predicted value . in order to evaluate the efficacy of tiotropium in early - stage of copd progression , we examined its effects on the rate of fev1 decline in copd patients with an fev170% . a retrospective cohort study was performed which included copd patients who were diagnosed between january 1 , 2004 , and july 31 , 2012 , at seoul national university hospital , seoul national university bundang hospital , and seoul metropolitan government - seoul national university boramae medical center . we classified patients into two groups , namely the tiotropium group and the control group , based on the use of tiotropium . the tiotropium group consisted of patients who had been prescribed the drug for more than 30 days during the follow - up period . the control group is defined as patients who had never received tiotropium or any muscarinic antagonist during the follow - up period . although patients who received any muscarinic antagonist were excluded , other copd medications were allowed . we compared the forced vital capacity ( fvc ) , and the yearly rates of fev1 decline , between groups . the protocol was approved by the ethical review committee of seoul national university hospital , seoul national university bundang hospital , and seoul metropolitan government - seoul national university boramae medical center ( irb number h-1302 - 002 - 459 ) . patients were aged 40 years with a post - bd fev170% of the predicted value and an fev1/fvc of less than 0.70 at initial point . all patients performed spirometry , including post - bd test , more than two times at a certain time of the year throughout the follow - up period . the following patients were excluded : those with asthma , lung cancer , or pulmonary tuberculosis , those that had previously undergone pulmonary resection , and those who were long - term users of short - acting muscarinic antagonist . information was collected on age , sex , body mass index , smoking history , comorbidities , symptoms , concomitant medication for copd , pre- and post - bd spirometry data , and radiographic findings . spirometry was performed according to the criteria of the american thoracic society.7 the longitudinal validity of the spirometers was also verified . the predicted values for fev1 and fvc were calculated using the methods of morris.8 initially , pre - bd spirometry was performed , followed by inhalation of salbutamol ( 200 g ) . propensity score matching was carried out to reduce potential confounding by nonrandom assignment or unbalanced covariates.9,10 the propensity score was calculated by logistic regression analysis using the following covariates : age , sex , height , weight , history of smoking , initial fev1 , presence of symptoms ( cough , sputum , or dyspnea ) , presence of emphysema , and use of any respiratory medications from the following list : inhaled corticosteroids ( icss ) , short - acting 2-agonists , long - acting 2-agonists ( labas ) , combinations of icss and labas , theophylline , or mucolytics . comparison of groups was performed using t - tests for normally distributed continuous data , the wilcoxon rank - sum test for non - normal continuous variables , and the chi - square test for categorical variables . the decline rate of post - bd fev1 , pre - bd fev1 , post - bd fvc , and pre - bd fvc over time was analyzed with random slope , random intercept mixed linear regression , adjusted for covariates of age , sex , body mass index , smoking status , and baseline fev1 and fvc.11 this statistical methodology is same as the one used in other landmark studies such as eclipse and torch study , which evaluated the rate of lung function decline in copd.12,13 in this model , fev1 or fvc changed linearly for each patient and the intercepts and slopes among patients were assumed to be random with covariance parameters . to investigate the impact of tiotropium further , subgroup analyses were conducted on patients who had received tiotropium for more than 50% of the medication possession ratio ( mpr ) . this was calculated by dividing the total number of prescription days by the total number of follow - up days . it is known that copd patients with emphysema decline rapidly.12 for this reason , subjects were classified according to the presence of emphysema , which was diagnosed using computed tomography imaging at the beginning of the follow - up . all statistical analyses were performed using stata version 12 ( stata corp . , college station , tx , usa ) . a retrospective cohort study was performed which included copd patients who were diagnosed between january 1 , 2004 , and july 31 , 2012 , at seoul national university hospital , seoul national university bundang hospital , and seoul metropolitan government - seoul national university boramae medical center . we classified patients into two groups , namely the tiotropium group and the control group , based on the use of tiotropium . the tiotropium group consisted of patients who had been prescribed the drug for more than 30 days during the follow - up period . the control group is defined as patients who had never received tiotropium or any muscarinic antagonist during the follow - up period . although patients who received any muscarinic antagonist were excluded , other copd medications were allowed . we compared the forced vital capacity ( fvc ) , and the yearly rates of fev1 decline , between groups . the protocol was approved by the ethical review committee of seoul national university hospital , seoul national university bundang hospital , and seoul metropolitan government - seoul national university boramae medical center ( irb number h-1302 - 002 - 459 ) . patients were aged 40 years with a post - bd fev170% of the predicted value and an fev1/fvc of less than 0.70 at initial point . all patients performed spirometry , including post - bd test , more than two times at a certain time of the year throughout the follow - up period . the following patients were excluded : those with asthma , lung cancer , or pulmonary tuberculosis , those that had previously undergone pulmonary resection , and those who were long - term users of short - acting muscarinic antagonist . information was collected on age , sex , body mass index , smoking history , comorbidities , symptoms , concomitant medication for copd , pre- and post - bd spirometry data , and radiographic findings . spirometry was performed according to the criteria of the american thoracic society.7 the longitudinal validity of the spirometers was also verified . the predicted values for fev1 and fvc were calculated using the methods of morris.8 initially , pre - bd spirometry was performed , followed by inhalation of salbutamol ( 200 g ) . propensity score matching was carried out to reduce potential confounding by nonrandom assignment or unbalanced covariates.9,10 the propensity score was calculated by logistic regression analysis using the following covariates : age , sex , height , weight , history of smoking , initial fev1 , presence of symptoms ( cough , sputum , or dyspnea ) , presence of emphysema , and use of any respiratory medications from the following list : inhaled corticosteroids ( icss ) , short - acting 2-agonists , long - acting 2-agonists ( labas ) , combinations of icss and labas , theophylline , or mucolytics . comparison of groups was performed using t - tests for normally distributed continuous data , the wilcoxon rank - sum test for non - normal continuous variables , and the chi - square test for categorical variables . the decline rate of post - bd fev1 , pre - bd fev1 , post - bd fvc , and pre - bd fvc over time was analyzed with random slope , random intercept mixed linear regression , adjusted for covariates of age , sex , body mass index , smoking status , and baseline fev1 and fvc.11 this statistical methodology is same as the one used in other landmark studies such as eclipse and torch study , which evaluated the rate of lung function decline in copd.12,13 in this model , fev1 or fvc changed linearly for each patient and the intercepts and slopes among patients were assumed to be random with covariance parameters . to investigate the impact of tiotropium further , subgroup analyses were conducted on patients who had received tiotropium for more than 50% of the medication possession ratio ( mpr ) . this was calculated by dividing the total number of prescription days by the total number of follow - up days . it is known that copd patients with emphysema decline rapidly.12 for this reason , subjects were classified according to the presence of emphysema , which was diagnosed using computed tomography imaging at the beginning of the follow - up . all statistical analyses were performed using stata version 12 ( stata corp . , college station , tx , usa ) . in a total of 587 patients , median spirometry measurements were taken three times annually , and the mean duration of follow - up was 38.9 months . of these patients , 257 received tiotropium . before propensity score matching , both the baseline characteristics and use of respiratory medications were significantly different between the tiotropium group and the control group ( table 1 ) . a greater proportion of patients in the tiotropium group than in the control group were current or former smokers ( 90.3% vs 84.8% ) . the mean pre- and post - bd fev1 was lower in the tiotropium group than in the control group ( pre - bd fev1 , 1.95 vs 2.24 l ; post - bd fev1 , 2.05 vs 2.34 l ) . moreover , a higher proportion of patients in the tiotropium group than in the control group were judged to have emphysema ( 72.1% vs 60.0% ) . seventy - five percent of patients in the tiotropium group reported respiratory symptoms , whereas only 61% of patients had respiratory symptoms in the control group . with regard to the use of respiratory medications other than tiotropium , patients in the tiotropium group were prescribed more than in the control group for the duration of the follow - up period . following propensity score matching , baseline characteristics , which showed differences between the two groups regarding smoking status , spirometry results , presence of emphysema , use of respiratory medication , and symptoms before propensity score matching ( table 1 ) , became similar after matching ( table 2 ) . there were no significant differences between the study groups regarding the rates of mean fev1 and fvc decline . specifically , the mean annual rate of decline in post - bd fev1 was 23.96.05 ml / yr in the tiotropium group and 22.55.89 ml / yr in the control group ( p=0.86 ) . the pre - bd fev1 decline was 30.46.2 ml / yr in the tiotropium group and 21.95.6 ml / yr in the control group ( p=0.31 ) . similarly , the mean annual rate of post - bd fvc decline was 55.18.61 ml / yr in the tiotropium group and 43.58.43 ml / yr in the control group ( p=0.33 ) . the annual rates of pre - bd fvc decline were 37.17.79 ml / yr in the tiotropium group and 33.37.45 ml / yr in the control group ( p=0.13 ) . in the subgroup analysis , among the 157 patients who received tiotropium for more than 50% of the mpr , the mean rate of decline in post - bd fev1 did not differ significantly between the tiotropium group and the control group ( 24.07.77 vs 27.04.71 ml / yr ; p=0.74 ) . additional propensity score matching was performed in patients with mpr 80% . after propensity score matching , 99 patients were included in long - acting muscarinic antagonist group , and 99 patients in control group . there were no significant differences between the two groups in the annual rate of decline in fev1 and fvc either before or after bd ( table s1 ) . although not statistically significant , a faster rate of fev1 decline was observed in patients with emphysema than in those without emphysema ( 29.2 vs 17.1 ml / yr ; p=0.154 ) . there were no significant differences between the two groups regarding the mean annual rate of post - bd fev1 decline . this was true both in patients with emphysema , and in those without emphysema ( patients with emphysema = 25.98.79 vs 32.08.76 ml / yr , p=0.622 ; patients without emphysema = 21.78.38 vs 14.07.97 ml / yr , p=0.545 ) . among patients who had not taken respiratory medication other than tiotropium during the follow - up period , the mean annual rates of pre- and post - bd fev1 decline were not significantly different between the two groups ( table 4 ) . in a total of 587 patients , median spirometry measurements were taken three times annually , and the mean duration of follow - up was 38.9 months . of these patients , 257 received tiotropium . before propensity score matching , both the baseline characteristics and use of respiratory medications were significantly different between the tiotropium group and the control group ( table 1 ) . a greater proportion of patients in the tiotropium group than in the control group were current or former smokers ( 90.3% vs 84.8% ) . the mean pre- and post - bd fev1 was lower in the tiotropium group than in the control group ( pre - bd fev1 , 1.95 vs 2.24 l ; post - bd fev1 , 2.05 vs 2.34 l ) . moreover , a higher proportion of patients in the tiotropium group than in the control group were judged to have emphysema ( 72.1% vs 60.0% ) . seventy - five percent of patients in the tiotropium group reported respiratory symptoms , whereas only 61% of patients had respiratory symptoms in the control group . with regard to the use of respiratory medications other than tiotropium , patients in the tiotropium group were prescribed more than in the control group for the duration of the follow - up period . following propensity score matching , baseline characteristics , which showed differences between the two groups regarding smoking status , spirometry results , presence of emphysema , use of respiratory medication , and symptoms before propensity score matching ( table 1 ) , became similar after matching ( table 2 ) . there were no significant differences between the study groups regarding the rates of mean fev1 and fvc decline . specifically , the mean annual rate of decline in post - bd fev1 was 23.96.05 ml / yr in the tiotropium group and 22.55.89 ml / yr in the control group ( p=0.86 ) . the pre - bd fev1 decline was 30.46.2 ml / yr in the tiotropium group and 21.95.6 ml / yr in the control group ( p=0.31 ) . similarly , the mean annual rate of post - bd fvc decline was 55.18.61 ml / yr in the tiotropium group and 43.58.43 ml / yr in the control group ( p=0.33 ) . the annual rates of pre - bd fvc decline were 37.17.79 ml / yr in the tiotropium group and 33.37.45 ml / yr in the control group ( p=0.13 ) . in the subgroup analysis , among the 157 patients who received tiotropium for more than 50% of the mpr , the mean rate of decline in post - bd fev1 did not differ significantly between the tiotropium group and the control group ( 24.07.77 vs 27.04.71 ml / yr ; p=0.74 ) . additional propensity score matching was performed in patients with mpr 80% . after propensity score matching , 99 patients were included in long - acting muscarinic antagonist group , and 99 patients in control group . there were no significant differences between the two groups in the annual rate of decline in fev1 and fvc either before or after bd ( table s1 ) . although not statistically significant , a faster rate of fev1 decline was observed in patients with emphysema than in those without emphysema ( 29.2 vs 17.1 ml / yr ; p=0.154 ) . there were no significant differences between the two groups regarding the mean annual rate of post - bd fev1 decline . this was true both in patients with emphysema , and in those without emphysema ( patients with emphysema = 25.98.79 vs 32.08.76 ml / yr , p=0.622 ; patients without emphysema = 21.78.38 vs 14.07.97 ml / yr , p=0.545 ) . among patients who had not taken respiratory medication other than tiotropium during the follow - up period , the mean annual rates of pre- and post - bd fev1 decline were not significantly different between the two groups ( table 4 ) . copd is characterized by airflow obstruction , which is usually degenerative , and has therefore been a therapeutic target . most clinical trials have included copd patients with an fev1 of less than 70% of the predicted value . although the combination of pulmonary rehabilitation plus pharmacotherapy reduced the decline of fev1,13,14 no single drug agent has been shown to conclusively reduce the progressive decline in lung function in moderate - to - severe copd . in this retrospective cohort study , we enrolled copd patients with an fev170% of the predicted value . in clinical practice , doctors usually prescribe drugs for symptomatic copd patients . hence , we found that tiotropium was prescribed more often for patients with such symptoms such as reduced lower lung function and emphysematous lung . these results are consistent with previous reports that symptomatic subjects require more respiratory care and indicate that there are several important physiological indicators of airflow obstruction severity , other than fev1.6,15,16 this fact leads to the difference in baseline characteristics observed here , which in turn causes selection bias . this was a real - world study , as opposed to a randomized controlled trial ( rct ) , and the absence of randomization clearly limits the reliability of conclusions from such studies . one of the most effective methods to control for equality between groups is propensity score matching.9 in order to reduce the selection bias , we adopted this approach . in this study , tiotropium did not reduce the annual rate of lung function decline in copd patients with fev170% . neither did tiotropium have any significant impact on the decline of pulmonary function in the subgroup analysis , which considered concomitant factors such as the presence of emphysema12,17,18 or prescription day . specifically , in a subgroup analysis in which patients who were followed up for more than 4 years were excluded , tiotropium did not reduce the annual rate of fev1 decline . however , when patients receiving respiratory treatment other than tiotropium were removed from the analysis , tiotropium did show a tendency to improve the annual rate of decline in pre - bd fev1 ( 24.08 ml / yr ) when compared with the control group ( 41.9 ml / yr , p=0.06 ) . although tiotropium did not significantly reduce the annual rate of fev1 decline in moderate - to - severe copd patients,3 the drug did seem to reduce said decline in a post hoc analysis of a patient subgroup with an fev1 between 50% and 70% of the predicted value.5 as the rate of fev1 decline has been reported to be faster in early - stage of copd than at later stages , one can speculate that it may be reduced by tiotropium in patients showing rapid fev1 decline . in fact , that is the reason we enrolled earlier stage of copd patients with an fev170% of the predicted value . unfortunately , we failed to demonstrate any effect of tiotropium on the prevention of disease progression in this study . in the uplift trial , which included patients with more severe copd than did this study , the decline in lung function averaged 41 ml / yr . a similar rate of fev1 decline was reported in the torch study.19 in contrast , the annual declines in fev1 were in the 2224 ml / yr range in this study . this is in accordance with the report that asian patients show a slower rate of decline ( 30 ml / yr ) than both western europeans ( 50 ml / yr ) and north americans ( 49.4 ml / yr).19 similarly , in the korean obstructive lung disease cohort , the mean annual rate of post - bd fev1 decline was 26.8 ml / yr in both stage 1 and stage 2 copd patients.20 this lower decline rate may cause difficulties in demonstrating any effect of tiotropium on lung function decline . taken together , this may indicate that our failure to demonstrate an effect of tiotropium was related to the fact that there was a low rate of fev1 decline in our study cohort . another possible explanation for the failure of tiotropium to reduce the rate of fev1 decline may be the concomitance of freely prescribed respiratory medications . there were no differences between our two groups with regard to the use of respiratory medications after propensity score matching . however , the proportion of patients who took respiratory medication other than tiotropium was lower in this study compared with that seen in the uplift trial . during the trial duration , 74% of patients in the uplift trial reported having taken ics , 72% labas , and 46% a fixed combination of the two.3 in our study , 2% of patients had taken ics , 10.4% labas , and 43.8% a combination of ics and labas . this discrepancy in respiratory medication may be responsible for the failure of tiotropium to reduce the rate of fev1 decline in this study , despite having been successful in the uplift study . this was because we included subjects only if they had performed spirometry regularly , at certain times of the year . another potential limitation is that patients with small exposure of tiotropium were included in this study . fifteen ( 5.8% ) patients were prescribed tiotropium for only 30 days and 103 ( 38.9% ) took it less than 50% of the time . among the subjects included in the propensity score analysis , 14 ( 6.9% ) patients had taken tiotropium for 30 days and 84 ( 41.6% ) took it less than 50% of the time . these patients could mask the effect of tiotropium on the rate in lung function decline . for confirmation , there was no significant difference between the two groups in the annual rate of decline in fev1 and fvc either before or after bronchodilation . in addition , due to the retrospective design , this study is subject to inherent selection bias . furthermore , this was a real - world study , in contrast to the uplift study , which was an rct . although rcts are essential research tools with a strong internal validity , their main limitation is their low generalizability to real - life conditions . thus , there is growing interest in real - world studies because of their close association with routine clinical practice . it is important to emphasize that the two approaches are complementary , so combining them increases their relative value and helps in overcoming their relative limitations . in conclusion , tiotropium does not reduce the rate of lung function decline in copd patients with an fev1 70% who were receiving other classes of respiratory medication during the study period . annual rates of fev1 and fvc decline before and after bronchodilation in patients with mp r 80% notes : control group was determined by propensity score matching . values for the rate of decline in fev1 and fvc are expressed as milliliters per year . p - value is adjusted for age , sex , bmi , smoking status , baseline fev1 , and fvc . abbreviations : fev1 , forced expiratory volume in 1 second ; fvc , forced vital capacity ; mpr , medication possession ratio ; se , standard error ; bmi , body mass index .

idiopathic pulmonary haemosiderosis ( iph ) is a rare cause of diffuse alveolar hemorrhage ( dah ) with unknown etiology . dah is characterized by haemoptysis , dyspnea , alveolar infiltrates on chest x - ray , and various degrees of anemia . in iph , it is classic disease of childhood , but approximately 20% of patients are presented during adulthood . radiographical manifestations of iph include patchy areas of opacities that usually involve both lungs and opacities similar to pulmonary edema . absolute diagnosis is based on the presence of hemosiderin - laden macrophages ( hlm ) without any evidence of pulmonary vasculitis , nonspecific granulomatous inflammation or deposition of immunoglobulins in respiratory secretion or biopsy materials . we reported the young adult man case because of 16-year period of iph disease ; responding to corticosteroid treatment very well and 8-year follow up after treatment . a 29-year old man was admitted to our clinic with complaints of fatigue , pallor , cough , bloody sputum , and shortness of breath in 2003 . in history , it showed a decrease in aeration and patchy areas of ground glass pattern in both lungs , especially in the lower lobes . a large number of hlm , interstitial edema and alveolar fibrosis , and foci of fresh hemorrhage were determined and he was diagnosed as idiopathic pulmonary hemosiderosis [ figure 1 ] . after blood transfusions , peripheral white cell count ( wbc ) and platelet ( plt ) count were normal , hemoglobin ( hb ) was 5.3 g / dl . serum biochemistry , ana , p - anca , c - anca , and rf were normal . erythrocyte sedimentation rate ( ers ) was 28 mm / h , serum iron was 18 mg / dl , iron binding capacity was 300 mg / dl . electrocardiography was normal , arterial blood gases were as follows : ph 7.4 , po266 , pco238 , sao294% . a large number of hemosiderin - laden macrophages and foci of fresh hemorrhage were determined . h and e , 100 and prussian blue , 100 two month later hemoptysis repeated and patient was admitted to our clinic . his and family history was not contributory . physical examination revealed pale skin and mucosa , other vital signs were normal . in respiratory system examination bilateral basal crackles were heard . a daily dose of 80 mg ( 1.5 mg / kg ) methylprednisolone was started , tapered to 48 mg / day 1 month later , and was stopped at the end of 6 month . the patient responded to treatment very well , and after that he had no complains . peripheral ground - glass appearance at the upper and middle lobe of right lung , and at the upper lobe of left lung were determined [ figure 2 ] . hb 13.2 g / dl , wbc , plt , and esr were normal and pulmonary function tests showed mild restriction . it has been 16 years from the begining of hemoptysis , 13 years from the diagnosis and 8 years from the treatment . since the beginning of treatment , there has been a very small amount of hemoptysis approximately less than 1 cc 1 - 2 times per year . peripheral ground - glass appearance at the upper and middle lobe of right lung , and at the upper lobe of left lung idiopathic pulmonary hemosiderosis is a rare disorder of unknown etiology and usually appears in children . the etiology of iph has been suspected to be autoimmune , allergic , genetic or environmental in different theories . some reports presenting that gluten - sensitive enteropathy and sensitivity to cow 's milk may accompany iph . our case was a young adult and such situations were not detected in our patients . findings of hemoptysis , iron deficiency anemia , shortness of breath , and parenchymal infiltration in chest radiography are typical as seen in many clinical articles . hrct appearances include patchy scattered areas of ground glass opacity and consolidation that usually involve the hilary and lower regions of the lungs , but involvement may be diffused . but in our case peripheral ground - glass appearance was presented at upper and middle lobe of right lung , and at the upper lobe of left lung . lung biopsy is necessary , the most important features are the presence of intact erythrocytes and numerous siderophages in the alveoli and the absence of any other pathologic sign like vascular malformation , immune deposition , capillaritis , vasculitis , granuloma , malignancy . in our patient , iph was diagnosed with the finding of hlm in lung biopsy material as in other studies . the use of systemic glucocorticoids for iph grew out of the impression of an immune pathogenesis . although the disease is rare , information from case reports and case series suggest that systemic glucocorticoids reduce the morbidity and mortality of acute episodes of alveolar bleeding and control progression to pulmonary fibrosis . the recommended dose was < 1 mg / kg / day prednisolone for two months period and then gradually reduced . in one study , we started daily dose of 80 mg ( 1.5 mg / kg ) treatment and tapered to 48 mg / day 1 month later , and stopped at end of 6 month . , we think that during childhood and young adult periods iph should be considered in patients presenting hemoptysis and anemia . for diagnosis tissue samples should be taken and as a treatment systemic corticosteroid therapy should be given .

measures of eeg during ect , for example , smaller post - seizure fractal dimension have predicted antidepressant response . the earlier study on this subject used bilateral ect . this study aims to examine if this finding holds good even for unilateral ect , using both fractal dimension and spectral power analysis of eeg . fifty - one right - handed , drug - free patients with major depressive disorder received right unilateral ects at 2.5 times their seizure threshold . a rater blind to the clinical data measured fractal dimension and spectral power of eeg during their second ect . depression was rated using hamiltons rating scale for depression at baseline and on the 3rd , 7th and 14th days following ect . seventeen patients reached criteria for early response of more than median percent improvement on hrsd on both 7th and 14th day . univariate analysis showed smaller fractal dimension and spectral power ( greater post - seizure eeg suppression ) in early responders compared to the late responders . effective seizure during unilateral ect may be characterized by high post - seizure eeg suppression .

non - melanoma skin cancer ( nmsc ) is a disease affecting mostly adult patients in their second half of life . forehead and temple are commonly affected by the major types of nmsc , i.e. basal cell carcinoma ( bcc ) and squamous cell carcinoma ( scc ) . the treatment of choice in particular for larger tumours is mohs surgery . in the case of deeper infiltration of the subcutaneous tissue , the preservation of the first branch of the facial nerve is not always possible to achieve a r0 resection ( complete removal with tumour - free excision margins ) . in these patients , upper lid ptosis and/or brow ptosis the correction of resulting functional impairment , especially of the lateral view , has priority . since most patients are elderly with significant dermoheliosis of facial skin , often dermatochalasis and significant comorbidities , the procedures should not add another significant risk . a 61-year - old male patient presented with a large and deep infiltrating bcc of the left temple . however , a combined brow and upper lid ptosis had developed and hampered his lateral view [ figure 1a ] . on the other hand ( e ) final wound closure of the direct brow lift and the upper lid blepharoplasty since he had a blepharochalasis , we used a combined approach , i.e. blepharoplasty of the upper lid and direct brow lift . the procedures were performed under local anaesthesia . because there was no prolapse of the adipose tissue , a simple skin resection of the upper lid skin was performed and sutured with 4 - 0 prolene ( ethicon , norderstedt , germany ) sutures . for the brow lift , an elliptical incision on the apical border of the brow was performed [ figure 1b ] . the height of the incision was measured before by pulling the brow to its intended height and marking the position . after the removal of skin , the deeper layers were carefully removed down to the insertion of the insertions of muscle fibres of the venter frontalis . the skin adjacent to the incision borders was loosened by blunt preparation to ensure a tension - free suturing . the frontalis muscle was resected and the ends were sutured by 4 - 0 pds ( ethicon ) sutures to position the brow [ figure 1c , d ] . wound closure was done by suturing the subcutaneous tissue with deep knots ( 4 - 0 pds ) and final wound closure with cutaneous 4 - 0 ethilon ( ethicon ) sutures [ figure 1e ] . after the removal of the suture , we recommended a preventive topical scar treatment ( cicaplast ; la roche posay , asnieres , france ) . an 86-year - old patient presented with a large scc in the left fronto - temporal region . four weeks later , she presented with a mild brow ptosis and an upper lid ptosis combined with blepharochalasis [ figure 2a c ] . ( a ) patient 2 after radical excision of a scc with upper lid and brow ptosis ( 1.8 mm in rest ) on the left side . ( b ) during upwards motion of the frontalis muscle , the left side does not improve . five days after bilateral upper lid blepharoplasty : frontal look with ( d ) closed eyes 1 week after surgery with sutures in place and ( e ) with open eyes demonstrate an acceptable aesthetic and functional outcome she did not want to have a correction of the brow but the upper lid . in this case , we performed blepharoplasty under local anaesthesia . after the resection of the upper lid skin with a monopolar radiofrequency tip ( ellman device [ ellmann int . , oceanside , ny ; usa ] , cut and coag mode ) , conservative fat pad removal was done by the incision of the medial and central septum . the patient was pleased with the outcome during the follow - up period of 2 years [ figures 2d e ] . a 61-year - old male patient presented with a large and deep infiltrating bcc of the left temple . however , a combined brow and upper lid ptosis had developed and hampered his lateral view [ figure 1a ] . on the other hand ( e ) final wound closure of the direct brow lift and the upper lid blepharoplasty since he had a blepharochalasis , we used a combined approach , i.e. blepharoplasty of the upper lid and direct brow lift . the procedures were performed under local anaesthesia . because there was no prolapse of the adipose tissue , a simple skin resection of the upper lid skin was performed and sutured with 4 - 0 prolene ( ethicon , norderstedt , germany ) sutures . for the brow lift , an elliptical incision on the apical border of the brow was performed [ figure 1b ] . the height of the incision was measured before by pulling the brow to its intended height and marking the position . after the removal of skin , the deeper layers were carefully removed down to the insertion of the insertions of muscle fibres of the venter frontalis . the skin adjacent to the incision borders was loosened by blunt preparation to ensure a tension - free suturing . the frontalis muscle was resected and the ends were sutured by 4 - 0 pds ( ethicon ) sutures to position the brow [ figure 1c , d ] . wound closure was done by suturing the subcutaneous tissue with deep knots ( 4 - 0 pds ) and final wound closure with cutaneous 4 - 0 ethilon ( ethicon ) sutures [ figure 1e ] . after the removal of the suture , we recommended a preventive topical scar treatment ( cicaplast ; la roche posay , asnieres , france ) . an 86-year - old patient presented with a large scc in the left fronto - temporal region . four weeks later , she presented with a mild brow ptosis and an upper lid ptosis combined with blepharochalasis [ figure 2a c ] . ( a ) patient 2 after radical excision of a scc with upper lid and brow ptosis ( 1.8 mm in rest ) on the left side . ( b ) during upwards motion of the frontalis muscle , the left side does not improve . five days after bilateral upper lid blepharoplasty : frontal look with ( d ) closed eyes 1 week after surgery with sutures in place and ( e ) with open eyes demonstrate an acceptable aesthetic and functional outcome she did not want to have a correction of the brow but the upper lid . in this case , we performed blepharoplasty under local anaesthesia . after the resection of the upper lid skin with a monopolar radiofrequency tip ( ellman device [ ellmann int . , oceanside , ny ; usa ] , cut and coag mode ) , conservative fat pad removal was done by the incision of the medial and central septum . the patient was pleased with the outcome during the follow - up period of 2 years [ figures 2d e ] . our department is a centre for dermato - oncology and dermato - surgery with inpatient and outpatient service and interdisciplinary tumour boards . careful surgical removal ensures a low rate of tumour recurrence and surgical complications . as long as the r0 resection is the primary goal of treatment , the preservation of facial ( or other ) nerves is not always possible . therefore , we need rehabilitative tools to deal with post - procedural palsy . in this report , we focussed on two procedures that are useful to correct upper lid and brow ptosis after nscm tumour surgery . the approaches aim to correct the resulting impairment , i.e. lateral or frontal limitations in eye view . since most of the patients are older than 60 years , often with significant comorbidities , the surgical approach has to respect this . aesthetic concerns are less important compared to the surgical correction of the ageing face without nmsc and palsy . there are a number of factors that need consideration when planning corrective surgery : age , ethnicity , skin type , forehead furrows , degree of visual impairment , and dermoheliosis . the use of simple but effective procedures that will not add an additional risk for the multi - morbid patients is our practical approach . we use a modification with suturing the frontalis muscle to obtain a significant lifting without placing any tension on the cutaneous sutures . a necessity of the technique is the careful avoidance of the dissection of the supraorbital neurovascular bundle . other adverse effects are rare and manageable . to ensure an aesthetic scar , preventive topical treatment by appropriate scar ointments or gels is recommended . the procedure can be combined with upper lid blepharoplasty to further improve the functional outcome . it has been suggested that patients with prominent brow fat pads will benefit from this technique . in a comparative study of brow lifting techniques , georgescu et al . observed a mean brow elevation of 1.74 mm using this method . in the particular situation , however , the limitations in the brow lift make this attempt less attractive for rehabilitative surgery . an endosopic brow lift results in greater amplitudes of brow elevation ( 3.44 mm ) . the procedure is more sophisticated than the direct brow lift but will be preferred in aesthetic indications . the direct brow lift , on the other hand , can also be employed for severe brow ptosis of > 4 mm . in some older patients , however , blepharoplasty alone can achieve a functional and aesthetic outcome that satisfies the patient without adding a surgical risk to his or her pre - existent comorbidities . from the functional point of view , upper lid blepharoplasty is not dependent on the resection of the orbicularis oculi muscle . recently , a less aggressive surgical approach to upper eyelid ptosis has been favoured for blepharoplasty . they way of dealing with post - surgical palsy has to be discussed with patients to identify their needs and complaints . keeping it simple often is a good compromise between the attempts of the surgeon to be ( almost ) perfect and the reality of the patient .

today , diabetes is considered as one of the most common metabolic disease worldwide and an important factor for death and disability in various countries including iran . world health organization predicted from 1997 to 2025 increasing of number of people with type ii diabetes worldwide 143 million to 300 million . although obesity and physical inactivity are most important risk factors for type 2 diabetes , but recent evidence suggests that oxidative stress play an important role in pathogenesis of this disease . increased insulin resistance and higher glucose and free fatty acids levels can increase the amount of active oxygen and generate oxidative stress in the body and activate pathways that are sensitive to stress[46 ] and following by it , free radicals impair insulin action through changes in the physical condition of target cell membrane.[79 ] on the other hand , non - enzymatic glycosylation reactions of proteins such as hemoglobin have been identified as major factors in the pathogenesis of chronic disorders of diabetes . antioxidants not only improve insulin secretion and performance through neutralizing free radicals , but also improve regulation of blood sugar and reduce complications of type ii diabetes and chronic disorders . also , inhibiting non - enzymatic glycosylation reactions of proteins can be effective on reducing chronic complications of diabetes disorders . fruits and vegetables are considered as rich resources in certain antioxidants , including carotenoids , vitamins a , c , e and some minerals such as selenium . according to a study by ceriello , taking the vitamins such as a , c and e reduce glycosylated hemoglobin and can thus be effective in reducing diabetes complications . according to another study by lupo , in parts of the world which mediterranean diet , including fruits and vegetables and oilseeds is high , the prevalence of type ii diabetes are less . in another study by laura franzin in 2008 , there was an inverse relationship between levels of plasma antioxidants and hba1c , in other words the levels of antioxidants were higher in patients who had glycosylated hemoglobin < 7 ( hba1c < 7 ) . because of high incidence of diabetes rising in iran and its effects on patients with compensated interminable and that seems one of the possible causes can make be through the antioxidant system deficiency ; hence the evaluation of relationship between antioxidant intake and levels of glycosylation reactions seems necessary . in addition , the effect of vegetables and fruits intake on lipids profiles , bun / creatinine and urine protein 24 h also is our study objective consideration . this cross - sectional study was performed in 2010 on 105 patients with type ii diabetes referred to the health center of khomeinishahr located in isfahan county in iran . initially , the profile patients including age , sex , education , occupation , smoking status , diabetes family history , age of diabetes onset , history of other diseases , drug consumption , height , weight , bmi and activity levels were recorded via questionnaire . before entering the study , subjects were given written informed consent and were described the aim and process of study and the individuals were asked if they wish to participate in the study . including criteria were diabetes , referring to khomeini shahr health centers in 2010 and were not pregnant . dietary data was obtained via a validated food frequency questionnaire ( ffq ) . to help people recall the exact amount of food eaten , we applied household utensils and cups and then for listed foods we used home scales manual and converted all of them to gram unit . for nutrient analysis , the amount of grams of food consumed was compiled by nutritionist 4 program . and for doing statistical analysis , spss software was compiled . to determine the correlation relationship between hemoglobin and independent variables pearson and spearman correlation coefficient was needed . to determine the severity of relationship between consumption of vegetables and fruits and glycosylated hemoglobin levels , linear regression was used . in this study , the mean consumption of fruits was 1.3 units and for consumed vegetables was 1.9 units , while the food pyramid recommended to get 2 - 4 units of fruits and 3 - 5 units of vegetables , and this indicated that the subjects do not receive at least average of fruits and vegetables recommended in food guide pyramid . in general , the distribution of total consumption of vegetables and fruits had a normal distribution . also the correlation between consumption of fruits and vegetables at the 0.05 significance level was observed [ table 1 ] . linear correlation between consumption of fruits and vegetables mean glycosylated hemoglobin ( hba1c ) was estimated 9.3 percent when people did not get fruits and vegetables . if fruit consumption is assumed constant , increasing consumption of 10 grams vegetables caused reducing 0.03 percent of glycosylated hemoglobin ( hba1c ) that this effect is borderline significant ( p = 0.049 ) , and if vegetables consumption assumed constant , increasing consumption of 10 grams fruits , caused reducing 0.02 percent of glycosylated hemoglobin but this effect was not significant . when in regression model , biased variables such as sex , age , drug consumption , diabetes type be added to the model , the effects of fruits and vegetables on glycosylated hemoglobin ( hba1c ) did not differ , but coefficients obtained in regression was not statistically significant . when the effect of both of fruits and vegetables consumption on glycosylated hemoglobin ( hba1c ) was considered , it can be said that by increasing consumption of 10 grams of fruit and vegetables , glycosylated hemoglobin ( hba1c ) significantly reduced 0.02 percent ( p = 0.014 ) [ table 2 ] . the relationship between hba1c levels and consumption of both of fruits and vegetables at the same time are shown in figure 1 . scatter plot to a and vegetables hba1c table 3 and table 4 show that there were no significant relationship between consumption of fruits and vegetables and lipids profiles , bun / creatinine and urine protein 24 h ( p > 0.05 ) . regression table , the relationship between consumption of fruits , vegetables and consumption of total vegetables and fruits with hba1c relationship between vegetables and fruits intake and bun / creatinine and urine protein 24 h relationship between vegetables and fruits intake and lipids profiles the aim of this study was to determine consumption of fruits and vegetables influence on the levels of glycosylated hemoglobin ( hba1c ) in type ii diabetic patients . various studies have shown that oxidative stress can play pathological role in many chronic diseases including obesity and diabetes . for example , according to a case - control study by ceriello , it was shown that serum antioxidant capacity reduced in diabetes patients . in another case - control study by goodarzi in iran show that plasma levels of malondialdehyde ( final product of fatty acids peroxidation due to increased free radicals ) in diabetic patients is higher than normal and significantly relationship between oxidative stress and blood glucose levels was demonstrated in this study . many clinical trial or laboratory studies show that supplementation with some antioxidants like vitamin e , vitamin c or alpha - lipoic acid ( ala ) could improve oxidative stress markers or levels of glycosylated hemoglobin ( hba1c ) in diabetic patients . for example , according to a study conducted by ceriello , glycosylated hemoglobin levels significantly reduced in group received vitamin e. in a cross - sectional study by sargeant on 2,678 men and 3,318 women , fruits , vegetables and other leafy vegetables consumption rate was evaluated with levels of glycosylated hemoglobin ( hba1c ) and showed that there is a significant relationship between receiving too much fruits or vegetables and glycosylated hemoglobin ( hba1c ) . although , hyperglycemia is involved as a primary factor in diabetes complications but it is still unclear whether path physiologic processes is identical in all diabetic complications or different mechanisms are involved in different organ diabetes complications . in general it can be said that oxidative stress and free radical production are involved as a hyperglycemia consequence of diabetes complications . there is evidence that rich of antioxidant diets reduced oxidative stress in patients with type ii diabetes . often it is observed in type ii diabetic or pre - diabetic patients , exogenous antioxidants compensate decreased plasma levels of plasma antioxidants . a recent hypothesis considered is that the consumption of vegetables and fruits that are rich of vitamins and other antioxidants can cause persistent high levels of antioxidants in diabetic patients . according to the results obtained in this study , it showed that consumption of fruits and vegetables can reduce hba1c levels and thus can be effective to reduce complications related to diabetes . the findings of previous study clearly indicate that hba1c is a useful biomarker of long - term glycemic control and can also predict lipid profile , thus , monitoring of glycemic control using hba1c could identify diabetic patients who are at a greater risk of cardiovascular complications.[2325 ] in spite of relationship between hba1c and lipid profile , the impact of consumption of fruits and vegetables on hba1c level was not the same on lipid profile , in this study . in general there was no significant relationship between consumption of fruits and vegetables and lipids profiles , bun / creatinine and urine protein 24 h. in this study , subjects did not receive least average of fruits and vegetables recommended in food pyramid and so should be advised to get more fruits and vegetables , also recommended to consider the impact of lifestyle factors in such studies .

following mastectomy , tissue expander reconstruction is one of the most common methods for breast reconstruction . the use of acellular dermal matrices ( adms ) has been popularized in the use of tissue expander reconstruction . this biologic matrix provides support in the lower pole of the breast and helps define the lower pole and inframammary fold . it also speeds the rate of expansion by reducing tension of the mastectomy skin and allows higher initial fill volumes . most recently , it has been suggested that adms help prevent capsular contracture , a common long - term problem with implants in the reconstructed breast [ 24 ] . although our institutional experience has been promising , other studies have suggested mixed , although generally positive , findings concerning the use of adms [ 3 , 5 ] . since clinical procedures are generally standardized , this suggests that the relative ability of the various adms to incorporate into soft tissues may relate to the manufacturing or processing of the particular material . bio - incompatibility due to lack of incorporation may cause seroma , infection , and reconstructive failure . the reconstructive process can be difficult enough for the patient without adding further cosmetic complications . structural support of the breast is necessary to provide the ideal breast shape and implant position . adms have been recognized as being beneficial by serving as a supplemental graft in breast reconstruction cases . they can supplement muscle coverage , shape the breast and inframammary fold , and possibly reduce the number of surgeries patients need to undergo . acellular dermal matrices are composed of the dermal layer and extracellular matrix of thin layers of donated skin that have had the epidermal layer removed . donor cellular material including major histocompatibility complex ( mhc ) proteins are removed in a series of further treatments to theoretically minimize immunological response in adm recipients . the ability of adms to promote significant revascularization and cellular infiltration make them an encouraging option for an array of tissue regeneration applications , including wound healing , soft tissue reconstruction , and augmentation [ 927 ] . a recently developed human adm produced by an anionic detergent and endonuclease - based decellularization process is a product trade - named dermacell ( lifenet health , virginia beach , va , usa ) . unique features of this allograft include removal of at least 97 % dna , an indication of complete decellularization and potentially reduced immunogenicity , terminally sterilization to a sterility assurance level of 10 , and being provided at room temperature and ready to use directly out of the package . here , we examine the post - mastectomy outcome of a two - stage breast reconstruction using this new human acellular dermal matrix , dermacell , in a 10 consecutive patient prospective cohort series . this prospective , consecutive cohort series included 10 female patients ranging in age from 33 to 59 years old who underwent mastectomies and presented to have breast reconstructions with the use of dermacell . of the 10 patients , eight had bilateral mastectomies and two had unilateral mastectomies , for a total of 18 breasts . one patient elected to have a prophylactic bilateral mastectomy after testing positive for the brca-1 gene . two patients underwent unilateral mastectomies , one of whom had previously undergone a unilateral mastectomy and reconstruction of the contralateral breast . the remaining patients each presented with breast cancer unilaterally and elected to have a mastectomy of the affected breast , as well as prophylactic mastectomy of the contralateral breast . two patients required neo - adjuvant chemotherapy , one required adjuvant chemotherapy , one additional patient required both neo - adjuvant and adjuvant chemotherapy , and two required radiation.table 1patient overviewpatient no.age ( years)post - op chemotherapyradiationuni- or bilateralduration of implant prior to 2nd stage ( weeks)expansion volumesmokerimplant volume150adj.nobilateral46400 ccno400 cc243nonobilateral24400 ccno600 cc352nonobilateral9380 ccno425 cc443neo - adj./adj.yesbilateraln / axyesx533nonobilateral7550 ccno500 cc656neo-adj.nobilateraln/axyesx759nonobilateral20700 ccno700 cc845neo-adj.yesunilateral ( r)41600 ccnoflap 948nonounilateral ( r)43220 ccno175 cc1033nonobilateral20500 ccno555 cc planned autologous reconstruction in a delayed fashion planned autologous reconstruction in a delayed fashion breast reconstruction was performed in two stages . in the first stage , following tissue expansion , patients advanced to the second stage of the procedure of immediate reconstruction for final placement of silicone implants . each reconstruction included placement of mentor cpx3 tall height contour profile implants ( mentor worldwide llc , santa barbara , ca , usa ) for tissue expansion and 16 6 cm dermacell patches , with one patient s dermacell patch tailored down to 12 6 cm . all expanders were placed with an initial volume of 100 cc intraoperatively using sterile saline . the pectoris muscle was elevated and the dermacell was sutured to the inferior portion of the muscle and to the inframammary fold to serve as a sling . a drain was placed in the breast pocket and in the subcutaneous pocket between the mastectomy skin and the dermacell . two weeks postoperative expansion began and continued between 7 and 46 weeks ( average 8 weeks ) until the desired volume . drains were removed between 6 and 21 days postoperatively ( average 15 days ) depending on their output . biopsy samples were taken from the patients and sent in formalin to dominion pathology laboratories ( norfolk , va , usa ) for sectioning and staining . hematoxylin and eosin ( h&e ) staining was undertaken to assess cellularity and general ultrastructure . additionally , immunohistochemical staining for the endothelial cell marker cd34 ( cd34 ) was used to assess vascularity and verhoef - von gieson ( vvg ) staining was performed to assess elastic fibers and general ultrastructure . histological assessments were determined by dermatopathologists kevagn fair , md ( dominion pathology ) and antoinette hood , md ( eastern virginia medical school , norfolk , va , usa ) . this prospective , consecutive cohort series included 10 female patients ranging in age from 33 to 59 years old who underwent mastectomies and presented to have breast reconstructions with the use of dermacell . of the 10 patients , eight had bilateral mastectomies and two had unilateral mastectomies , for a total of 18 breasts . one patient elected to have a prophylactic bilateral mastectomy after testing positive for the brca-1 gene . two patients underwent unilateral mastectomies , one of whom had previously undergone a unilateral mastectomy and reconstruction of the contralateral breast . the remaining patients each presented with breast cancer unilaterally and elected to have a mastectomy of the affected breast , as well as prophylactic mastectomy of the contralateral breast . two patients required neo - adjuvant chemotherapy , one required adjuvant chemotherapy , one additional patient required both neo - adjuvant and adjuvant chemotherapy , and two required radiation.table 1patient overviewpatient no.age ( years)post - op chemotherapyradiationuni- or bilateralduration of implant prior to 2nd stage ( weeks)expansion volumesmokerimplant volume150adj.nobilateral46400 ccno400 cc243nonobilateral24400 ccno600 cc352nonobilateral9380 ccno425 cc443neo - adj./adj.yesbilateraln / axyesx533nonobilateral7550 ccno500 cc656neo-adj.nobilateraln/axyesx759nonobilateral20700 ccno700 cc845neo-adj.yesunilateral ( r)41600 ccnoflap 948nonounilateral ( r)43220 ccno175 cc1033nonobilateral20500 ccno555 cc planned autologous reconstruction in a delayed fashion planned autologous reconstruction in a delayed fashion breast reconstruction was performed in two stages . in the first stage , following tissue expansion , patients advanced to the second stage of the procedure of immediate reconstruction for final placement of silicone implants . each reconstruction included placement of mentor cpx3 tall height contour profile implants ( mentor worldwide llc , santa barbara , ca , usa ) for tissue expansion and 16 6 cm dermacell patches , with one patient s dermacell patch tailored down to 12 6 cm . all expanders were placed with an initial volume of 100 cc intraoperatively using sterile saline . the pectoris muscle was elevated and the dermacell was sutured to the inferior portion of the muscle and to the inframammary fold to serve as a sling . a drain was placed in the breast pocket and in the subcutaneous pocket between the mastectomy skin and the dermacell . two weeks postoperative expansion began and continued between 7 and 46 weeks ( average 8 weeks ) until the desired volume . drains were removed between 6 and 21 days postoperatively ( average 15 days ) depending on their output . biopsy samples were taken from the patients and sent in formalin to dominion pathology laboratories ( norfolk , va , usa ) for sectioning and staining . hematoxylin and eosin ( h&e ) staining was undertaken to assess cellularity and general ultrastructure . additionally , immunohistochemical staining for the endothelial cell marker cd34 ( cd34 ) was used to assess vascularity and verhoef - von gieson ( vvg ) staining was performed to assess elastic fibers and general ultrastructure . histological assessments were determined by dermatopathologists kevagn fair , md ( dominion pathology ) and antoinette hood , md ( eastern virginia medical school , norfolk , va , usa ) . eight ( 15 breasts ) of the 10 breast reconstruction procedures were successfully completed and only minor complications were reported in two patients ( table 2 ) . major complications were seen in the two patients with failed breast reconstructions due to smoking and infection . patient outcomes were as follows ( table 2 ) : four breasts developed seromas , there were two surgical site infections , four experienced delayed healing , and three had flap necrosis . of particular note , there was no observation of hematomas or red breast syndrome in any patient . over half of the observed complications were limited to a single patient , limiting the complicative profile and associating patient comorbidities as the major contributor . it appears that a history of smoking caused mastectomy skin necrosis and a port - a - cath wound infection contributed to the failed reconstruction . this patient developed seromas of both breasts , a purulent right breast infection , and mastectomy flap necrosis of both breasts . this patient also had postoperative radiation and significant skin necrosis , apparently due to the radiation . the other patient who failed reconstruction developed a seroma , surgical site infection , flap necrosis , and experienced delayed healing of the left breast . these complications were most likely the result of the patient continuing to smoke throughout her postoperative recovery . the expanders were removed and the reconstruction was aborted.table 2patient outcomespatient no.seromahematomasurgical site infectionred breastdelayed healingflap necrosiscompleted reconstructionfailed reconstruction10/20/20/20/20/20/22/20/220/20/20/20/20/20/22/20/230/20/20/20/20/20/22/20/242/20/21/20/22/22/20/22/251/20/20/20/20/20/22/20/261/20/21/20/21/21/20/22/270/20/20/20/20/20/22/20/280/10/10/10/10/10/11/10/190/10/10/10/11/10/11/10/1100/20/20/20/20/20/22/20/2total4/180/182/180/184/183/1815/184/18 six patients that completed reconstruction did not experience any complications . 1 ) was one of these six patients that had a successful reconstruction with no infections , seromas , or other possible complications . she was 43 years old , had neoadjuvant chemotherapy for a left locally advanced breast cancer , and was advised to have a mastectomy . the patient also requested a contralateral prophylactic mastectomy with immediate reconstruction of both breasts . from histological analysis ( fig . 2 ) , it is evident that the dermacell had incorporated into the host tissue and allowed for revascularization . 1 a , b patient 2 preoperative before undergoing bilateral mastectomy with immediate reconstruction . c , d patient 2 postoperative 7 months after placement of permanent implantfig . 2 a , b h&e stain of dermacell sample taken from patient 2 s left breast at 7 months postoperative . viewed under 100 . the upper arrows identify blood vessels while the lower arrows identify fibroblasts a , b patient 2 preoperative before undergoing bilateral mastectomy with immediate reconstruction . c , d patient 2 postoperative 7 months after placement of permanent implant a , b h&e stain of dermacell sample taken from patient 2 s left breast at 7 months postoperative . the upper arrows identify blood vessels while the lower arrows identify fibroblasts another notable completed reconstruction was performed on patient 5 ( fig . she was 33 years old , had a strong family history of breast cancer , and therefore decided to be tested for the brca-1 gene mutation . the patient tested positive for the mutation and elected to undergo prophylactic mastectomy of both breasts with immediate breast reconstruction . a seroma developed on the left breast between the skin and the dermacell on day 10 . a drain was placed between the skin and dermacell , and was then removed 1 week later . the patient had expansion for about 7 weeks and then returned for tissue expander removal and placement of the permanent implants . at this time , the dermacell was found to be incorporated into the surrounding tissues.fig . 3 a patient 5 preoperative before undergoing bilateral mastectomy with immediate reconstruction . b patient 5 postoperative 1 month after exchange of tissue expander for implant a patient 5 preoperative before undergoing bilateral mastectomy with immediate reconstruction . b patient 5 postoperative 1 month after exchange of tissue expander for implant patient 10 , one of the eight patients with completed reconstruction , was a 33-year old who originally presented for breast augmentation ( fig . , she was found to have left breast cancer and was counseled to undergo mastectomy . the patient agreed to have left breast mastectomy and also opted to have prophylactic right breast mastectomy followed by immediate breast reconstruction . after expansion continued to the desired volume , the tissue expander implants were removed at 20 weeks and replaced with permanent silicone breast implants . during placement of the permanent implants , it was noted that the dermacell matrix had incorporated into the surrounding tissue and showed signs of granulation budding ( fig . 5).fig . 4 a patient 10 preoperative before undergoing bilateral mastectomy with immediate reconstruction . b patient 10 postoperative 7 months after mastectomy and placement of tissue expanders and dermacellfig . 5patient 10 , 5 months after expansion of the dermacell , the matrix is incorporated into the surrounding tissue and show signs of granulation a patient 10 preoperative before undergoing bilateral mastectomy with immediate reconstruction . b patient 10 postoperative 7 months after mastectomy and placement of tissue expanders and dermacell patient 10 , 5 months after expansion of the dermacell , the matrix is incorporated into the surrounding tissue and show signs of granulation histology analysis confirms the incorporation of the dermacell matrix into the surrounding tissue , aiding in reconstruction . observations for all biopsy samples included presence of fibroblasts , intact ultrastructure including elastin , and vasculature ( figs . rarely , a foreign body response was observed and was noticeable at the same position as polarizable material . the foreign body response was consistent with suture material , as evidenced by the regular pattern and minimum associated inflammation . pseudo - capsule formation was generally seen on the side of the implant facing the expander , which occurred as a benign response to the expander material . the opposite interface between the implant and the host tissue exhibited some tissue integration and minimal inflammation , which was consistent with normal healing . the implant material looked more organized with less vasculature and fewer living cells compared to host tissue . these findings are consistent for a stable material being slowly incorporated and remodeled after a few weeks to a few months after surgery when biopsy samples were taken.fig . 6hematoxylin and eosin staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . note the intact ultrastructure and also evidence of cellular in - growth as apparent fibroblasts ( arrows ) at 10 magnificationfig . 7cd34 staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . evidence of robust vascularization is noted by reddish - brown stains apparently associated with blood vessels ( arrows ) at 10 magnificationfig . 8verhoef - vangeisen staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . note abundance of elastin ( arrows ) in this 10 magnification hematoxylin and eosin staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . note the intact ultrastructure and also evidence of cellular in - growth as apparent fibroblasts ( arrows ) at 10 magnification cd34 staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . evidence of robust vascularization is noted by reddish - brown stains apparently associated with blood vessels ( arrows ) at 10 magnification verhoef - vangeisen staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . eight ( 15 breasts ) of the 10 breast reconstruction procedures were successfully completed and only minor complications were reported in two patients ( table 2 ) . major complications were seen in the two patients with failed breast reconstructions due to smoking and infection . patient outcomes were as follows ( table 2 ) : four breasts developed seromas , there were two surgical site infections , four experienced delayed healing , and three had flap necrosis . of particular note , there was no observation of hematomas or red breast syndrome in any patient . over half of the observed complications were limited to a single patient , limiting the complicative profile and associating patient comorbidities as the major contributor . it appears that a history of smoking caused mastectomy skin necrosis and a port - a - cath wound infection contributed to the failed reconstruction . this patient developed seromas of both breasts , a purulent right breast infection , and mastectomy flap necrosis of both breasts . this patient also had postoperative radiation and significant skin necrosis , apparently due to the radiation . the other patient who failed reconstruction developed a seroma , surgical site infection , flap necrosis , and experienced delayed healing of the left breast . these complications were most likely the result of the patient continuing to smoke throughout her postoperative recovery . the expanders were removed and the reconstruction was aborted.table 2patient outcomespatient no.seromahematomasurgical site infectionred breastdelayed healingflap necrosiscompleted reconstructionfailed reconstruction10/20/20/20/20/20/22/20/220/20/20/20/20/20/22/20/230/20/20/20/20/20/22/20/242/20/21/20/22/22/20/22/251/20/20/20/20/20/22/20/261/20/21/20/21/21/20/22/270/20/20/20/20/20/22/20/280/10/10/10/10/10/11/10/190/10/10/10/11/10/11/10/1100/20/20/20/20/20/22/20/2total4/180/182/180/184/183/1815/184/18 six patients that completed reconstruction did not experience any complications . 1 ) was one of these six patients that had a successful reconstruction with no infections , seromas , or other possible complications . she was 43 years old , had neoadjuvant chemotherapy for a left locally advanced breast cancer , and was advised to have a mastectomy . the patient also requested a contralateral prophylactic mastectomy with immediate reconstruction of both breasts . from histological analysis ( fig . 2 ) , it is evident that the dermacell had incorporated into the host tissue and allowed for revascularization . 1 a , b patient 2 preoperative before undergoing bilateral mastectomy with immediate reconstruction . c , d patient 2 postoperative 7 months after placement of permanent implantfig . 2 a , b h&e stain of dermacell sample taken from patient 2 s left breast at 7 months postoperative . viewed under 100 . the upper arrows identify blood vessels while the lower arrows identify fibroblasts a , b patient 2 preoperative before undergoing bilateral mastectomy with immediate reconstruction . c , d patient 2 postoperative 7 months after placement of permanent implant a , b h&e stain of dermacell sample taken from patient 2 s left breast at 7 months postoperative . the upper arrows identify blood vessels while the lower arrows identify fibroblasts another notable completed reconstruction was performed on patient 5 ( fig . she was 33 years old , had a strong family history of breast cancer , and therefore decided to be tested for the brca-1 gene mutation . the patient tested positive for the mutation and elected to undergo prophylactic mastectomy of both breasts with immediate breast reconstruction . a seroma developed on the left breast between the skin and the dermacell on day 10 . a drain was placed between the skin and dermacell , and was then removed 1 week later . the patient had expansion for about 7 weeks and then returned for tissue expander removal and placement of the permanent implants . at this time , the dermacell was found to be incorporated into the surrounding tissues.fig . 3 a patient 5 preoperative before undergoing bilateral mastectomy with immediate reconstruction . b patient 5 postoperative 1 month after exchange of tissue expander for implant a patient 5 preoperative before undergoing bilateral mastectomy with immediate reconstruction . b patient 5 postoperative 1 month after exchange of tissue expander for implant patient 10 , one of the eight patients with completed reconstruction , was a 33-year old who originally presented for breast augmentation ( fig . , she was found to have left breast cancer and was counseled to undergo mastectomy . the patient agreed to have left breast mastectomy and also opted to have prophylactic right breast mastectomy followed by immediate breast reconstruction . after expansion continued to the desired volume , the tissue expander implants were removed at 20 weeks and replaced with permanent silicone breast implants . during placement of the permanent implants , it was noted that the dermacell matrix had incorporated into the surrounding tissue and showed signs of granulation budding ( fig . 5).fig . 4 a patient 10 preoperative before undergoing bilateral mastectomy with immediate reconstruction . b patient 10 postoperative 7 months after mastectomy and placement of tissue expanders and dermacellfig . 5patient 10 , 5 months after expansion of the dermacell , the matrix is incorporated into the surrounding tissue and show signs of granulation a patient 10 preoperative before undergoing bilateral mastectomy with immediate reconstruction . b patient 10 postoperative 7 months after mastectomy and placement of tissue expanders and dermacell patient 10 , 5 months after expansion of the dermacell , the matrix is incorporated into the surrounding tissue and show signs of granulation histology analysis confirms the incorporation of the dermacell matrix into the surrounding tissue , aiding in reconstruction . observations for all biopsy samples included presence of fibroblasts , intact ultrastructure including elastin , and vasculature ( figs . rarely , a foreign body response was observed and was noticeable at the same position as polarizable material . the foreign body response was consistent with suture material , as evidenced by the regular pattern and minimum associated inflammation . pseudo - capsule formation was generally seen on the side of the implant facing the expander , which occurred as a benign response to the expander material . the opposite interface between the implant and the host tissue exhibited some tissue integration and minimal inflammation , which was consistent with normal healing . the implant material looked more organized with less vasculature and fewer living cells compared to host tissue . these findings are consistent for a stable material being slowly incorporated and remodeled after a few weeks to a few months after surgery when biopsy samples were taken.fig . 6hematoxylin and eosin staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . note the intact ultrastructure and also evidence of cellular in - growth as apparent fibroblasts ( arrows ) at 10 magnificationfig . 7cd34 staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . evidence of robust vascularization is noted by reddish - brown stains apparently associated with blood vessels ( arrows ) at 10 magnificationfig . 8verhoef - vangeisen staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . note abundance of elastin ( arrows ) in this 10 magnification hematoxylin and eosin staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . note the intact ultrastructure and also evidence of cellular in - growth as apparent fibroblasts ( arrows ) at 10 magnification cd34 staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . evidence of robust vascularization is noted by reddish - brown stains apparently associated with blood vessels ( arrows ) at 10 magnification verhoef - vangeisen staining of biopsy from patient # 1 following 16 weeks in situ placement of acellular dermal matrix . this was not unexpected as anthony et al . noted a trend towards significance in their study on the incidence of complications in breast reconstruction using acellular dermal matrices . . demonstrated a significant complication rate for smokers in their retrospective study of hundreds of breast reconstruction patients . other studies have also noted an increased incidence of complications of patients who smoke after post - mastectomy breast reconstructions [ 31 , 32 ] . in addition to the immediate advantages of providing structural support , the ultimate goal of using adm s is to enable integration of the matrix into the host tissue . the two blinded dermatopathologists , who assessed dermacell samples , identified numbers of blood vessels and fibroblasts that support incorporation into the host cell . a similar case series was recently published where dermacell was used for two - stage breast reconstructions in 10 breast cancer patients . after excluding one patient for tobacco use , the author found comparable results with 14 out of 17 breasts successfully reconstructed to the second stage implant sequence . there were few complications reported , including the noteworthy absence of red breast syndrome in all patients , and most postoperative complications occurred in a patient who relapsed into tobacco use . histological analysis of biopsy samples taken of the adm from eight patients indicated incorporation with minimal inflammation and normal healing , consistent with our findings . another study using in vivo rat models compared several different adm s , including dermacell . 7 postoperative , the dermacell matrices had nearly twice the number of blood vessels as the next highest adm . in regards to recellularization , it was found that dermacell had the statistically significant highest amount of cell density at days 7 , 14 , and 21 postoperative as well as the greatest amount of cell infiltration . the success reported here with dermacell may be due to the processing and sterility of the material . the process involves the use of a solution of non - denaturing anionic detergent ( n - laurol sarcosinate ) , recombinant endonuclease ( benzonase ( merck kgaa , darmstadt , germany ) , and antibiotics ( polymixin b , vancomycin , and lincomycin ) , resulting in a material that exhibits at least 97 % nucleic acid removal , while maintaining biomechanical strength . dermacell allografts are preserved in glycerol , stored at room temperature , and are ready to use without needing to be rehydrated . the allografts are terminally sterilized at ultra - low temperatures to achieve a 10 sterility assurance level [ 35 , 36 ] . the temperature of irradiation has proved to be important because sterilizing grafts at very low temperatures minimizes free radicals and eventual tissue damage [ 37 , 38 ] . in addition to the temperature at which irradiation occurs , the solutions used to clean the human tissue can determine the quality of the allograft . when harsh chemicals such as acetone , sodium hydroxide , and hydrogen peroxide are used , they can greatly damage the soft tissue . many factors are involved in the processing of a human tissue allograft with a sterility assurance level of 10 . an allograft with a sterility assurance level of 10 is equivalent to the level of sterility provided with implantable medical devices , and the use of such sterile allografts could result in a lower rate of infection than when using autografts obtained from a second procedural site [ 40 , 41 ] . by using low doses of gamma irradiation at very low temperatures , this process is able to ensure a high degree of safety without compromising biological and biomechanical properties [ 4246 ] . the decellularization process used to prepare dermacell is designed to remove cellular elements while retaining matrix structure , theoretically yielding a material with reduced antigenicity , increased biocompatibility , and subsequently reduced complications . ( lifecell corporation , bridgewater , nj , usa ) , a decellularized human dermal matrix , is aseptically processed and freeze - dried . it is treated with a buffered salt solution to separate and eliminate the epidermis , then washed with a series of mild non - denaturing detergent solutions to solubilize and eliminate all cells . alloderm undergoes a sterilization process that includes electron beam irradiation , which provides for a sterility assurance level of 10 . flexhd ( musculoskeletal transplant foundation , edison , new jersey , usa ) is human allograft skin that is minimally processed to remove epidermal and dermal cells , a process that maintains the integrity of the matrix . it is packaged in an ethanol solution and must be soaked in a sterile solution before implantation . the allograft tissue complies with the requirements of united states pharmacopeia ( usp ) < 71 > sterility tests but is not terminally sterilized , and thus claims no level of sterility assurance . while all of these products are acellular dermal matrices , there are many differences between the three , including the degree of sterilization . dermacell maintains a sterility assurance level of 10 , which assures that no more than one in a million implants could potentially be infected with microorganisms . in comparison , alloderm is only aseptic with a sterility assurance level of 10 and , in theory , about one in one thousand implants could potentially be infected . flexhd is not terminally sterilized , but meets the requirements of usp < 71 > sterility tests . the same sterility standards are not followed by all organizations that supply bone and tissue allografts . since there is no standard definition of sterile , there can be clinical concern and confusion regarding infection . contaminated allografts can cause significant complications or even death in patients who receive the infected tissue . in addition to compromising the health of patients , surgical site infections due to contaminated allografts can place a notable financial burden on patients , hospitals , and health care providers . as determined by the centers for medicare and medicaid services ( cms ) , surgical site infections are considered to be preventable complications and hospitals will not be reimbursed for the care of patients suffering from such complications . in 2005 , a study performed in pennsylvania reported that the average charge for patients that developed a hospital infection ( $ 173,206 ) was about four times as high as the charge for patients with the same diagnosis who did not contract an infection ( $ 44,367 ) . therefore , allograft - associated infections place additional financial burden on patients and have a significant economic impact on hospitals . rigorous screening , in combination with stringent aseptic tissue processing followed by terminal sterilization , can help eliminate allograft - associated infections . decellularization of human allograft tissue performed during tissue processing results in a material devoid of immunogenic components , theoretically allowing for improved graft biocompatibility , incorporation , and healing . the decellularization process used in the treatment of dermacell results in at least a 97 % reduction in the dna content of the tissue , reduced , in one analysis , to approximately 15.97 ng / mg dry weight . the residual dna value for alloderm is much higher at 272.8 ng / mg dry weight . the presence of erythema of mastectomy skin flaps following matrix implant , also known as red breast syndrome , has been reported when using either human or xenograft matrices [ 20 , 53 , 54 ] . while not fully understood , it is suggested that this may be an inflammatory response to the implanted matrix or preservatives [ 20 , 53 ] . it is of special interest to note that there were no cases of red breast syndrome in this case series using dermacell . while no generalizable conclusions can be reached from this small patient population , the processing of dermacell adm resulting in significant reduction in dna and cellular content may result in the absence of erythema . to compare the effectiveness of different acellular dermal matrices , histological analysis was performed on biopsies taken as part of this cohort and from additional two - stage breast reconstruction patients , also treated by this study author . one patient had received alloderm , while flexhd was used in the second patient . at 6 weeks the revascularization of the dermacell tissue indicates that incorporation has occurred . in comparison , the alloderm tissue biopsy ( fig . 10 ) did not have nearly as many blood vessels and fibroblasts as the dermacell tissue . the arrow on the left identifies a blood vessel and the arrow on the right identifies a fibroblastfig . the upper arrow identifies a hematoma and the lower arrow identifies a fibroblast six - week postoperative biopsy for dermacell . the upper arrow identifies a fibroblast and the lower arrow identifies a blood vessel postoperative biopsy for alloderm . the arrow on the left identifies a blood vessel and the arrow on the right identifies a fibroblast postoperative biopsy for flexhd . the upper arrow identifies a hematoma and the lower arrow identifies a fibroblast it is important for successful breast reconstructions utilizing acellular dermal matrices to facilitate the incorporation of the host tissue into the allograft material . acellular dermal matrices are able to support larger and faster tissue expansion , reduce capsular contracture , and decrease the rate of revision [ 5558 ] . the ability of dermacell to incorporate into host tissue with good revascularization and recellularization could provide for quicker breast reconstructions with fewer complications . overall , patients that experienced the most complications postoperatively were those with unhealthy lifestyle behaviors , most notably smoking . the results of our case series suggest that dermacell is at least comparable to other adms , within the limitations of the small cohort size .

mucogingival problems form a definitive diagnosis that includes an array of clinical findings , namely gingival recession ( gr ) , shallow vestibule , inadequate width of attached gingiva ( ag ) and aberrant frenulum . surgical endeavor by goldman for the correction of three specific problems , namely periodontal pockets that extend beyond the ag reaching the alveolar mucosa , an abnormal traction of the frenulum that can transmit tension for the gingival margins and cause recessions , and the functional condition of a shallow vestibule that promotes a decrease of the ag levels , initiated the era of mucogingival surgery that has motivated other clinicians to develop numerous refinements . several surgical procedures have then been described in continuation by various clinicians for these aforementioned mucogingival entities . these surgical solutions , although possessing their own merits , posed certain difficulties ; mainly , their inadequacy to treat other problems simultaneously . a single - step solution to mucogingival problems by a surgical intervention , i.e. bridge flap procedure , is the continuation of the same endeavor . the following case series represents a cost - effective single - step entity to correct multiple mucogingival problems at a time with less morbidity to donor tissue and to evaluate the correction gained by this bridge flap procedure . after clearance from local ethical committee , 18 teeth with recession in eight otherwise systemically healthy non - smoker patients in the age group of 2040 years , who came with the chief complaint of poor esthetics in the lower anteriors due to recession and/or hypersensitivity , were selected for the procedure after phase one therapy . clinical examination revealed an inadequate zone of ag ( tension test - positive ) with either shallow vestibule or aberrant frenum attachment in all cases [ figure 1 and 2 ] . clinical parameters recorded using unc-15/who periodontal probe at base line and 9 months after procedure included probing pocket depth ( ppd distance from gingival margin to base of pocket ) , gr ( distance from cementoenamel junction to gingival margin ) , width of ag ( distance from base of pocket to mucogingival junction ) , and clinical attachment level ( cal distance from cementoenamel junction to base of pocket ) , as shown in tables 13 ] . the procedure was well explained to the patient and informed consent was obtained for surgery . pre - operative photograph showing insufficient width of attached gingiva , presence of diastema , pronounced labial inclination , and miller 's class iii recession on teeth 31 and 41 pre - operative photograph showing recession on teeth 31 and 41 along with insufficient attached gingiva and high frenum attachment pre - operative ( at base line ) post - operative ( at 9 months ) change in pre- and post - operative values the surgical procedure included the bridge flap technique as introduced by marggraf and later on modified by romanos . the technique included an arch - shaped incision in the vestibule at approximately 2 gr + 2 mm from the gingival margin . an incision into the perisoteum was placed at its base and the bone was exposed so that scar formation could occur . a spilt - thickness flap was elevated in the apicocoronal direction by making a sulcular incision , connecting it with the first incision so that the whole bridge flap could be elevated and repositioned coronally to cover the denuded root surfaces . the repositioned flap was then pressed for 3 min and independent sling sutures were placed [ figure 3 and 4 ] . written post - operative instructions were given to the patients and they were prescribed analgesic ( ibuprofen 400 mg twice daily ) for 3 days . sutures were removed 10 days after the surgical procedure and patients were asked to maintain meticulous oral hygiene . all surgical procedures were performed by the same clinician and both pre - operative and post - operative measurements were recorded by the same individual . intra - operative photograph showing arc - shaped incision and bridge flap repositioned coronally with independent sling sutures intra - operative photograph showing arc - shaped incision and bridge flap repositioned coronally with independent sling sutures t test . for the purpose of analysis , statistical package for social sciences version 15.0 was used . results showed statistically significant root coverage and significant gain in ag and clinical attachment with average value of 1.778 1.003 mm , 3.527 0.915 mm , and 1.945 0.265 mm , respectively . highest gain in ag was 4.6 mm , whereas the least gain was 1.8 mm , and the highest recession coverage came to an average of 87.5% , whereas the least was 25% . results also showed non - significant changes in periodontal ppd . while reviewing the literature , we came across a number of articles addressing mucogingival problems and their surgical solutions . however , an unresolved controversy still exists in the literature regarding the adequate ag for periodontal health maintenance , and the contemporary opinion suggests that the regions with < 2 mm ag and thin gingival tissue are at increased risk of gr even if it is possible to maintain the gingival health in the areas with insufficient or absent ag . hence , mucogingival therapy should be advocated for gingival augmentation and to create adequate vestibular depth in areas with insufficient ag.[58 ] contrary to the reports of marggraff , romanos et al . , and vijaylaxmi et al . , who all stressed very little on the gain of width of ag by this particular technique , we observed a surprising and highly statistically significant gain . the highest gain of ag width in our study was 4.6-times of the pre - operative , and the least was 1.8-times , with an average gain of 3.5 mm [ figure 5 and 6 ] . we selected our cases regardless of the etiology of gr mainly on the patients complaint of esthetics and hypersensitivity . but , on close survey of our cases selected , anatomical variations for gr , viz . high frenum pull , shallow vestibule , thin gingival tissue or insufficient width of ag , were present in one or the other patient as contributing factors for gr . by achieving such a significant gain in the apico - coronal dimension of ag , we at least could abolish the aforementioned contributing etiologic factors for gr , and the incidence of recurrent recession was also decreased considerably by the simultaneous extension of the vestibule . the mean percentage gain in cal was around 41% in our study , which also re - asserts the gain in width of ag by this surgical endeavour . the mean percentage change in ppd from the mean pre - operative value had been only 14.3% , clinically not much reduction could have been possible as the cases selected were already scaled and root planed thoroughly . post - operative photograph ( 9 months ) . marked increase in width of the attached gingiva can be appreciated post - operative photograph ( 9 months ) the mean root coverage obtained in our study was 55% and , contrary to the study of vijaylakshmi et al . and romanos et al . , we could not achieve any 100% root coverage . this dissimilarity of our results could be because of us being more liberal in our case selections in terms of diastema presence , pronounced labial inclinations , shallow vestibule , or frenum pull at the same time . the least coverage attained in our study was 25% and , therefore , the range of root coverage ( 2587.5% ) is similar to the range of root coverage obtained by any root coverage procedure . the minimum values of root coverage , i.e. 25% , in few cases could be due to the very thin gingiva present , which posed great difficulties in coronally repositioning the bridge flap that is a partial thickness flap . to overcome this difficulty , modifications such as combination of connective tissue grafts along with this bridge flap technique can be advocated . we kept our study cases limited to the lower anteriors to get unbiased results as well as to be able to treat multiple mucogingival problems at the same time . in terms of root coverage , it is highly important to have a proper case selection , e.g. presence of diastema , improper proximal contacts , thinness of gingival , or pronounced labial inclinations , which , otherwise , significantly lowers the outcome of root coverage . the results of our cases where open proximal contacts were present are contrary to the results shown by vijaylakshmi et al . based on our results , we also recommend that a completely preserved interdental septum is a conditio sine qua non as advocated by marggraf . the present case series presents a cost - effective single - step entity to correct multiple mucogingival problems at a time with less morbidity to donor tissue ; however , lack of inclusion control groups are its limitation and , hence , further controlled randomized clinical trials are required . several surgical techniques are described in the literature to correct mucogingival problems . the surgical endeavor , bridge flap procedure , gave a successful solution by attaining root coverage , increasing the width of ag , deepening the vestibule to a desired extent , and simultaneously correcting the problem of high frenum if present , in one step only , along with gain in cal and reduction of ppd .

in this work , we coupled on - line kinetic capillary electrophoresis with mass spectrometry ( kce - ms ) to study conformational dynamics of dna g - quadruplexes in solution . we showed that peaks shift and its widening in kce can be used for measuring rate and equilibrium constants for dna metal affinity interactions and g - quadruplex formation ; and ion mobility mass spectrometry ( im - ms ) provided information about relative sizes , absolute molecular masses and stoichiometry of dna complexes . we did not expect such a big difference in migration time between folded and unfolded dna . our initial guess was that we could measure only a thermodynamic folding constant , but not rate constants for the fast conformational change . we believe that kce - ms will be used complementally to cd , sf , and spr techniques for studying nucleic acid structures and functions , screening dna / rna binding compounds and selecting aptamers . this work was supported by the natural sciences and engineering research council of canada ( grant rgpin/385739 - 2010 ) and canada foundation for innovation ( project no .

in recent years , manganese oxides ( mno2 ) have attracted considerable interests due to their distinctive physical and chemical properties and wide applications in catalysis , ion exchange , molecular adsorption , biosensor , and energy storage [ 1 - 5 ] . specifically , manganese oxides have been extensively evaluated as electrode materials for supercapacitors due to their low cost and environmental benignity compared to noble metal oxides such as ruo2[6 - 8 ] . in the development of supercapacitors , nanostructured electrode materials have received great interests as they exhibit higher specific capacitance and rate capability compared to traditional bulk materials . over the years , various nanostructured manganese oxides , including one - dimensional ( 1-d ) ( nanorodes , nanowires , nanobelts , nanoneedles , and nanotubes ) , two - dimensional ( 2-d ) ( nanosheets , nanoflakes ) , and three - dimentional ( 3-d ) ( nanospheres , nanoflowers , hollow urchins ) nanostructures , have been synthesized [ 9 - 16 ] . 3-d hierarchical porous structures often produce more active sites and exhibit more favorable electrochemical properties than 2-d and 1-d structures . however , facile synthesis and mass production of complex 3-d nanostructures are still a challenge in the areas of materials science [ 17 - 20 ] . it has been reported that a core - shell structure with spherically aligned nanorods of -mno2 can be prepared through a simple room temperature reaction between mnso4 and ( nh4)2s2o8 with a catalyst of ag in an acid solution . a similar method used by gong et al . is able to synthesize mno2 hollow urchins with a reactive template of carbon spheres . wang et al . also reported the synthesis of hierarchical -mno2 spheres by the reaction between mnso4 and k2s2o8 with the addition of cuso4 in an acidic solution . however , the preparation of 3-d nanostructured mno2 in the thin film form has never been reported . since the use of composite electrodes introduces additional undesirable interfaces in the electrode material with the risk of negating the benefits of electrochemistry using nanostructures , thin film electrodes enable us to investigate the electrochemical properties of the active material itself without the influence of binders and conductive additives as required for composite electrodes . in this paper , we propose the stratagem to synthesize 3-d -mno2dandelion - like spheres and 2-d -mno2nanoflakes by a reaction between mnso4and ( nh4)2s2o8 in a na2so4solution at low temperatures . with a platinum ( pt ) substrate submerged into the reaction solution , the nanostructured mno2can the effects of the synthesis temperature on the morphology of the films are investigated , and the capacitive behaviors of nanostructured mno2thin films with different morphologies are studied and compared . a typical synthesis of nanostructured mno2was performed by dissolving mnso4 , ( nh4)2s2o8 , and na2so4with a molar ratio of 1:1:1 in 30 ml deionized water at room temperature . the concentrations of mn , s2o8 , and so4 in the solution are the same as 0.1 mol l. a pt substrate was submerged into the solution , while the solution was magnetically stirred in a beaker at room temperature ( rt ) for 12 h or at 80 c for 2 h. one side of the pt substrate was covered with parafilm , so that mno2can only be deposited on one side . after the reaction , the pt substrate was washed using distilled water and then dried in the vacuum at 60 c overnight . structure and crystallinity of thin films were characterized using a shimadzu xrd-6000 x - ray diffractometer with cu k radiation at a scanning rate of 1min . surface morphology of the as - deposited thin films was characterized using a hitachi s-4100 field emission scanning electron microscope ( fesem ) . weights of the mno2thin films were measured using a microbalance with an accuracy of 0.01 mg . all electrochemical measurements were conducted using a solartron 1287 electrochemical interface combined with a solatron 1260 frequency response analyzer . for the electrochemical measurements , a three - electrode cell system composed of a mno2thin film electrode as the working electrode , a high surface carbon rod as the counter electrode , and an ag / agcl reference electrode was employed . the capacitive behaviors of the as - deposited mno2thin films were characterized by cyclic voltammetry ( cv ) in 1 m na2so4electrolyte at room temperature . cv measurements were performed on the three - electrode cells in the voltage window between 0 and 0.9 v at different scan rates from 20 to 200 mv s. electrochemical impedance spectra ( eis ) of different thin film electrodes were measured at the open - circuit potential in the frequency range from 100 khz to 10 mhz . a typical synthesis of nanostructured mno2was performed by dissolving mnso4 , ( nh4)2s2o8 , and na2so4with a molar ratio of 1:1:1 in 30 ml deionized water at room temperature . the concentrations of mn , s2o8 , and so4 in the solution are the same as 0.1 mol l. a pt substrate was submerged into the solution , while the solution was magnetically stirred in a beaker at room temperature ( rt ) for 12 h or at 80 c for 2 h. one side of the pt substrate was covered with parafilm , so that mno2can only be deposited on one side . after the reaction , the pt substrate was washed using distilled water and then dried in the vacuum at 60 c overnight . structure and crystallinity of thin films were characterized using a shimadzu xrd-6000 x - ray diffractometer with cu k radiation at a scanning rate of 1min . surface morphology of the as - deposited thin films was characterized using a hitachi s-4100 field emission scanning electron microscope ( fesem ) . weights of the mno2thin films were measured using a microbalance with an accuracy of 0.01 mg . all electrochemical measurements were conducted using a solartron 1287 electrochemical interface combined with a solatron 1260 frequency response analyzer . for the electrochemical measurements , a three - electrode cell system composed of a mno2thin film electrode as the working electrode , a high surface carbon rod as the counter electrode , and an ag / agcl reference electrode was employed . the capacitive behaviors of the as - deposited mno2thin films were characterized by cyclic voltammetry ( cv ) in 1 m na2so4electrolyte at room temperature . cv measurements were performed on the three - electrode cells in the voltage window between 0 and 0.9 v at different scan rates from 20 to 200 mv s. electrochemical impedance spectra ( eis ) of different thin film electrodes were measured at the open - circuit potential in the frequency range from 100 khz to 10 mhz . figure 1a , b shows the xrd patterns of the mno2thin films synthesized at different temperatures . 1a , b can be indexed as a tetragonal symmetry of -mno2with a space group of i4/m ( jcpds card , no . 1a are sharper and stronger , indicating that the degree of crystallinity of the products is enhanced as the synthesis temperature increases . however , some small impurity peaks observed from both fig . 1a , b can be indexed as mn3o4 , which is probably due to the incomplete oxidation reaction between mnand s2o8 . the complete formation of mno2from the solution can be expressed as the following reaction:(1 ) axrd pattern of the mno2thin film synthesized at rt , bxrd pattern of the mno2thin film synthesized at 80 c ( thedotted linesinredcolor represent the diffraction peaks from mn3o4 ) , andcphotos of the pt substrate before and after the deposition -mno2 has usually been found to be the product of the oxidation of mn by s2o8 either through a hydrothermal reaction or through a mild solution reaction [ 21 - 23 ] . it has been observed in this study that the formation of nanostructured mno2 is preferred to deposit on the pt substrate rather than in the solution . 1c ) in this study is quite simple and convenient compared with electrochemical deposition , which is usually employed to prepare mno2 thin films . the morphologies of the mno2 thin films synthesized at different temperatures are shown in fig . it can be seen from fig . 2a that the film synthesized at rt is composed of uniform microscopic spheres with diameters ranging from 0.5 to 1 m . the magnified fesem image ( fig . 2b ) shows that these microscopic spheres are composed of nanowhiskers , resulting in a dandelion - like morphology . reported that a core - shell structure microspheres ( 23 m ) of -mno2 can be obtained by the reaction between mnso4 and ( nh4)2s2o8 with the addition of agno3 catalyst at rt for 12 days . the thin shell of this structure is composed of nanorods , and this morphology can only be obtained with the existence of ag . wang et al . reported that sea urchin - shaped microspheres ( ~1 m ) of -mno2 can be obtained by the reaction between mnso4 and k2s2o8 with the addition of cuso4 at 70 c for 3 days . in the present work , the 3-d hierarchical structure of -mno2 can be obtained at rt in a relatively short synthesis time ( 12 h ) without using any agno3 or cuso4 since the addition of catalyst of agno3 or cuso4 may induce ag or cu impurities in the final mno2 products . it is assumed that reaction between mn and s2o8 in the na2so4 solution with a pt substrate is relatively faster compared to the previous studies . it is interesting to observe a morphology evolution of the film , as the synthesis temperature increases . as shown in fig . 2d , the film synthesized at 80 c exhibits another type of porous structure with nanoflakes almost vertically aligned on the pt substrate . the magnified fesem image ( fig . 2e ) shows that the average size of the nanoflakes is about 500 nm , and the thickness of the nanoflakes is less than 50 nm . fesem images for rt synthesized mno2:alow magnification andbhigh magnification , cschematic illustration for the possible formation mechanism of dandelion - like mno2microspheres , fesem images for 80 c synthesized mno2:dlow magnification andehigh magnification , andfschematic illustration for the possible formation mechanism of mno2nanoflakes the possible formation mechanism for the hierarchical mno2 spheres is schematically illustrated in fig . , the crystal growth process always includes two steps : the initial nucleation stage and following crystal growth stage . after which , the absorbed mno2 colloids on the pt substrate tend to aggregate loosely to form spherical appearance due to their high surface energies . because the reaction temperature is at rt , gibbs energy for nucleation of new mno2 sites is low . as a consequence , mno2 colloids tend to attach on the habit planes of existing mno2 sites , leading to the formation of 1-d nanowhiskers from the initial colloidal microspheres . with increase in the processing duration , finally dandelion - like 3-d microspheres of mno2 on the pt substrate appear . however , on the contrary to wang s finding , the increase in reaction temperature in this study is unable to improve the formation of 3-d hierarchical microspheres of mno2 but leads to the formation of 2-d nanoflakes . this phenomenon can be explained by the change in growth mechanisms as shown in fig . when the reaction temperature is increased to 80 c , the reaction rate is greatly enhanced along with the high rate of adsorption of mno2 colloids to the pt substrate . under such circumstances , in addition to the one - dimentional growth of the nuclei along the low energy direction , the growth of the nuclei along other directions can also happen due to the fast nucleation and adsorption rates of mno2 at an elevated temperature . the 3-d and 2-d nanostructured mno2 are promising as electrode materials for supercapacitors due to their porous structure , large surface area , and short diffusion length for protons or alkali cations . in order to evaluate the electrochemical properties of the as - deposited thin films , the thin film samples with different nanostructures were characterized in aqueous 0.1 m na2so4 electrolyte with cv measurements . the cv curves at various scan rates from 20 to 200 mv s for the sample a and b are shown in fig . 3a , b ( sample a represents mno2 with dandelion - like sphere morphology , and sample b represents mno2 nanoflakes ) . it can be seen that the curves at the low scan rate of 20 mv s for both samples exhibit ideal symmetrical rectangle - like shape indicating ideal capacitive behavior . as the scan rate increases , slight distortion from the ideal symmetrical rectangle shape however , it is clear to see that the distortion from the rectangularity of the cv curves for the sample a is much less than that for the sample b , indicating much better rate performance of the sample a due to its finer nano - architecture . the specific capacitance of the nanostructured mno2 film can be obtained by the following equation:(2 ) where q is the voltammetric charge , e is the voltage window ( 0.9 v ) , and m is the mass of the active material of the electrode . the specific capacitances at different scan rates for both samples are shown in fig . s , the sample a exhibits a much higher specific capacitance of about 230 f / g than 180 f / g of the sample b. as the scan rate increases , the specific capacitance for both samples decreases , which is typical for electrochemically active mno2 materials . at the highest scan rate of 200 mv s , the sample a can maintain 66% of its full capacitance ( we set the specific capacitance at 20 mv s as the full capacitance ) while the sample b can only maintain 55% of its full capacitance , indicating that the sample a has much better rate capability than that of the sample b. figure 3d compares the nyquist plots for the mno2 thin films with different nanostructures . a depressed semicircle in the high - frequency range corresponding to the charge - transfer resistance , and a straight sloping line in the low - frequency range corresponding to the diffusive resistance can be observed for both samples . as shown in fig . 3d , it is clear that the sample a has lower charge - transfer resistance and diffusive resistance compared with those of the sample b , confirming the superior capacitive behavior of the sample a. based on the previous paper by chou et al . , surface area of thin film can be derived from the double layer capacitance by fitting eis using equivalent circuit . the calculated surface areas of the sample a and the sample b are about 414 and 125 m g respectively . pseudocapacitance of mno2 mainly originates from the adsorption of cations in the electrolyte ( m = li , na , and k ) on the surface of mno2 and also possible intercalation / deintercalation of h and alkaline metal cations in the bulk of mno2 . since the 3-d dandelion - like microspheres of the sample a are composed of much finer nanowhiskers with very small size , they provide a much larger surface area per gram and shorter diffusion length for cations , comparing to the relatively large 2-d nanoflakes of the sample b. therefore , the morphology advantage of sample a explains why it can exhibit a higher specific capacitance and better rate capability than those of the sample b. athe cv curves at various scan rates from 20 to 200 mv sfor the mno2film synthesized at rt , bthe cv curves at various scan rates from 20 to 200 mv sfor the mno2film synthesized at 80 c , cspecific capacitance at various scan rates for the mno2thin films with different nanostructures , anddeis spectra of mno2thin films with different nanostructures the sample a , showing better capacitive behavior in rate capability test , was further investigated for long - term cycling stability . the cv curves at different cycling stages and variation of specific capacitance over 2,000 cycles are shown in fig . 4a , the cv curves for the 1st , 500th , 1000th , and 2000th cycles almost overlapped with each other , indicating excellent cycling stability . after 2,000 cycles , there is no degradation of the capacitive behavior , indicating no significant structural or microstructural changes in the mno2thin film electrodes . the cv curve after 2,000 cycles is noted to become more symmetrical with the rectangular shape compared with the first cycle , indicating improved capacitive behavior after long time cycling . 4b ) slightly decreases for the first 3040 cycles then starts to increase very slowly with the cycling . as shown in the xrd results , there is a small amount of mn3o4exist in the film . mn with a lower oxidation state in the mn3o4is probably oxidized to mnduring the long time cv cycling , resulting in improved capacitive behavior and an small increase of specific capacitance . acv curves for the 1st , 500th , 1,000th , and 2,000th cycles for the mno2thin film synthesized at rt andbthe variation of specific capacitance with respect to cycle number for the mno2thin film synthesized at rt mno2thin films with nanostructures have been prepared on pt substrates by a facile and mild solution method . the mno2film prepared at rt with a long reaction time is composed of dandelion - like microspheres , which consists of nanowhiskers with very small size . the reaction temperature plays an important role in controlling the surface morphology of the film . as the reaction temperature was increased to 80 c , a film composed of nanoflakes can be prepared in a very short time . the cv measurements indicate that mno2thin films prepared by this method are promising as electrodes for supercapacitors . the film composed of dandelion - like microspheres exhibited a higher specific capacitance and better rate capability than the film composed of nanoflakes , which is probably due to the high surface area and smaller feature of the microspheres . the excellent cycling stability and good rate capability of the film composed of dandelion - like microspheres coupled with the simple and low cost synthesis method make this material attractive for large applications . this research is supported by national university of singapore and agency for science , technology and research through the research grant r-265 - 000 - 292 - 305 ( 072 134 0051 ) .

10% of the population has experienced some kind of dental trauma , of which 0.5 to 16% were cases of avulsion of permanent teeth . once avulsed , the tooth must be replanted into its own socket3 in order to reestablish functional normality . for success of replantation , maintenance of viable periodontal ligament cells present on root surface hence , immediate replantation23,4,1516 ( within 15 minutes after avulsion ) or storage of the exarticulated tooth in solutions compatible with cell viability until replantation3 are critical procedures . given that the 7 - 11-year - old age group is the most commonly affected age range5 , school professionals should be aware of the importance of immediate treatment procedures in cases of tooth avulsion20 . the findings of previous study of our research team20 conducted in the city of adamantina , so paulo state , brazil , have shown unawareness of school professionals with respect to emergency procedures for management of avulsed permanent teeth . however , this is not an isolated finding . therefore , there is an oncoming need to establish standard operational procedures about initial treatment of tooth avulsion , in order to increase the prognosis of the affected teeth1,1012,20,2425 . the purpose of this study was to evaluate the efficacy of an informative campaign about how to proceed in cases of dental trauma applied to urban elementary school professionals from the city of adamantina , brazil . the informative campaign on tooth avulsion was directed to teachers and employees of elementary schools at the city of adamantina , so paulo state , brazil . these professional deal with children in the 7 - 12-year - old age range . the campaign was based on use of banners and folders with the heading " save a tooth " , which contained information on what to do in cases of tooth avulsion . the material had simple wording and figures to facilitate understanding by all readers ( figure 1 ) and was placed on the walls and distributed to teachers and employees of the schools . participants were oriented about immediate replantation , appropriate washing of avulsed tooth , with emphasis on extra care not to friction the root surface , storage of the avulsed tooth in milk and referral to the dentist as soon as possible for replantation and follow - up . twelve of the 19 urban schools existing in adamantina were visited and had access to folders , posters and lectures . the campaign reached a total of 120 teachers and school staff . in a previous study conducted in schools in adamantina20 , 117 teachers and school staff answered a questionnaire for evaluation of their knowledge on tooth avulsion . all participants who had answered the questionnaire before the campaign had access to this information . six months after the campaign , the participants were asked to answer to a questionnaire - based survey in order to evaluate the efficacy of the campaign . the questionnaire had to be returned within 7 days with no mention of participant 's identity . part i consisted of general information , including age , gender , educational level and time at present job . part ii included notions of basic emergency procedures , experience with tooth avulsion , knowledge of handling accidental situations , and knowledge of specific initial procedures in case of tooth avulsion ( figure 2 ) . this questionnaire was based on preexisting models1,1012,20 . as there is no standard questionnaire validated for such purpose , after the questionnaires had been returned , the answers were counted and the percentages for each question were calculated . data obtained in this study were compared to some data of the previous study conducted in adamantina20 . the following questions were statistically compared : importance of emergency management of tooth avulsion , storage media employed for avulsed teeth and the most frequently indicated storage medium . statistical analysis was performed with epi info software version 6.04 using the proportion test at 5% significance level . most participants were females ( 93.75% ) between 41 - 60 years old ( 53.75% ) with a college degree ( 77.5% ) and time at work ranging from 1 - 5 years ( 22.5% ) . when asked if they had experience with dental traumatisms , only 13.75% answered yes , and 100% of the participants considered emergency procedures to be mandatory in such cases . in case of a student getting hurt , 42.5% said they would seek help from the school dentist , 31.25% would seek a dentist in town and 15% would call the student 's parents . only 6.25% said they would seek treatment at dental schools and 3.75% would seek the local health system . the questions regarding tooth avulsion revealed that only 2.5% of school professionals would replant an avulsed tooth . of the 80 participants , 42.5% would wash the avulsed tooth ; of these , 44% would use milk and 38.6% would use water . for 81.25% of the participants , the avulsed tooth should be kept in a storage medium if replantation could not be performed immediately . moreover , of this total , 84.61% would place the tooth in milk , 3.06% in water , 1.53% in saliva and 7.66% would keep the tooth involved in paper or plastic ( dry ) ( figure 4 ) . the answers to part iii of the survey demonstrated that the campaign was well - accepted by the participants . only 11.25% reported not to have read the informative posters and 3.25% had not attended the seminars . professionals who did not know about the campaign either missed work on a seminar day or did not receive the folders . participants of the seminars ( 96.25% ) and readers of the posters and folders ( 88.75% ) considered the campaign as a positive and clarifying resource and approved the accomplishment of similar campaigns in other schools . comparison of data to those of the previous study conducted in adamantina20 pointed to the efficacy of the campaign . the statistical analysis revealed statistically significant difference between data , as presented in figure 5 . it is known that when replantation is performed immediately after tooth avulsion ( up to 15 minutes)23,5,16 or when the tooth is stored in appropriate solutions compatible with cell survival69,1415,17,19,2123,2627 , case prognosis is greatly enhanced . when immediate replantation is impossible , storage of the tooth in a humid environment is mandatory3 . milk has been indicated as the preferred storage medium for avulsed teeth in several countries . in addition to being readily available , it preserves cell viability for up to 6 hours9,22 . keeping the tooth dry compromises the periodontal ligament cells and children between 7 and 11 years of age are the most commonly affected by dental trauma5 . therefore , professionals that work with children must be aware of the importance of emergency treatment and how to proceed in cases of tooth avulsion . mori et al.20 conducted a study in adamantina six months before the campaign and observed that only 7.6% of school professionals would place the tooth in milk for transportation to the dental office . in addition to the lack of knowledge , other investigators1,1012,20,2425 have also reported the importance of a massive awareness of the population about dental trauma , usually by means of informative campaigns . nonetheless , the spread of information about emergency procedures in cases of dental trauma is often needed . in the present study , a campaign for providing information about dental trauma to school professionals was performed in adamantina , so paulo state , brazil . the purpose of the campaign was to alert school professionals on the importance of immediate treatment procedures in case of tooth avulsion and instruct them on how to handle these situations . the results of the survey , applied 6 months after the campaign , revealed that the professionals realized how crucial immediate treatment is for cases of tooth avulsion , as 100% of the participants answered positively to this question . we noted that after the campaign , a larger number of professionals ( 92.34% ) said they would store the avulsed tooth in some storage medium ; milk was the preferred solution ( 84.61% ) . in a previous study conducted with the same population , only 42% would place the tooth in some kind of liquid , and milk would be used in only 7.6% of the cases20 . the results of that study20 could be compared to the data collected after completion of the campaign , confirming its efficacy ( figure 3 ) . the results were positive after the campaign due to the promotion of awareness of school professionals . the informative folders , posters and lectures achieved their goal , namely to widen the knowledge of these professionals on tooth avulsion . thus , the use of information in a clear and simple manner is one of the best ways to improve treatment success of avulsed permanent teeth . these results confirm the efficacy of the campaign , which was considered approved by all participants . this suggests that accomplishment of informative campaigns about tooth avulsion in schools is a valid alternative and can be extended to the general population to further increase success rates of treatment of tooth avulsion and consequently promote oral health .

routine vl testing is not yet widely available globally , with some countries still not recommending routine vl testing at all in their national guidelines , or recommending it only in the case of suspected treatment failure ( mdecins sans frontires [ msf ] access campaign : http://msfaccess.org/hiv-hcv-diagnostic-product-guide-2015 ) . the latest implementation data show access to vl testing to be highly variable mostly severely limited with considerable variations in the approaches to monitoring encountered ( msf access campaign ) [ 13 , 14 ] . information compiled from the international association of providers of aids care database show that although 47 of 54 lmics have guidelines in line with latest who recommendations to ensure routine vl testing , the reality is that only a handful are implementing vl testing ( msf access campaign : http://msfaccess.org/hiv-hcv-diagnostic-product-guide-2015 ) . six countries recommend vl testing only in the case of suspected treatment failure : mandatory in morocco , myanmar , and south sudan ( if the test is available ) , and optional in haiti , india , and zimbabwe . india and zimbabwe only recommend targeted vl testing and it is only optional to confirm treatment failure . most countries are also still recommending routine immunological treatment monitoring , with only 8 countries reporting that they have dropped routine cd4 testing post art initiation ( cameroon , kenya , malawi , namibia , south africa , swaziland , thailand , and uganda ) . a recent survey by who targeting 122 lmics found that only 20% of art patients receive vl testing . in the lmics surveyed , there were only 2 vl instruments , on average , per 8706 people on art , with 10% of these machines not in operation because they had not yet been installed or required repair , or due to lack of reagents and the absence of staff training . findings from an in - depth qualitative survey of experts based in india , kenya , malawi , south africa , and zimbabwe ( march may 2014 ) found that in malawi , with > 400 000 patients on art , only 37 000 received a vl test in 1 year ( 2013 data ) . this suggests that vl implementation currently falls well short of guidelines and limits the public health impact of these programs [ 1 , 2 ] . in addition , insufficient and overburdened healthcare professionals , poor training and lack of knowledge , and weak transport and laboratory systems were all considered barriers to scale - up of vl testing [ 11 , 1618 ] . furthermore , access to testing is higher in central and urban areas compared with more remote areas [ 16 , 17 ] . lmics , however , have strong ambitions to expand vl testing , with many countries having scale - up plans in place . these countries face immense funding and implementation hurdles that may mean it is hard to put new guidelines into practice . many of these same barriers were documented with cd4 and early infant diagnostic ( eid ) scale - up , and those involved in vl scale - up should consider the lessons learned [ 1619 ] . countries were able to improve coverage using various models , such as the hub - and - spoke system . although there has been significant expansion in cd4 and eid capacity in resource - poor settings , this does not always translate to access , particularly for lower - level health facilities . also , turn - around time and loss of samples or results remain problematic , and more work is needed in this area . several promising laboratory - based vl technologies and simple poc tests are expected to become available soon for use in lmics [ 5 , 12 , 21 ] . poc testing will not necessarily be a magic bullet and will most likely complement not replace conventional testing platforms . one of the key problems with current poc tests is that most of them are still plasma based for vl testing , which is inconvenient due to the continued need for trained phlebotomists and electrical centrifuges for plasma separation , both limited in lmics . poc implementation will still require training of staff , implementation of quality control mechanisms , and operational research to assess poc performance and the benefits of poc testing over and above conventional testing . context - based decisions will need to be made in terms of whether to use decentralized poc tests , or centralized dbs - based laboratory tests . one survey found that across lmics , vl laboratory - based technology performed an average of 5 tests ( range , 1191 ) per machine per day , despite most platforms having a nominal capacity of at least 90 tests per day , suggesting that vl technology faces the double challenge of limited numbers of vl tests due to high reagent costs and costs per test being considerably higher than they would be if platforms were efficiently used . decisions on where to place vl testing must take several factors into account , including patient volume and characteristics ( eg , to what degree transient , remote , or stigmatized populations are linked to the healthcare system ) ; cost per test and anticipated levels of instrument usage , which can yield different cost efficiencies ; human resource skill level and task - shifting policies and consequences ; and the time to a results - based intervention . throughput needs should also take into account likely scale - up of diagnostics in the immediate future . laboratory - based vl testing may make more sense , for example , in high - prevalence settings with existing sample transport systems , particularly if people are already enrolled in care and the aim of the vl test is to confirm that treatment is working effectively . in other contexts , poc testing will be an important catalyst in promoting adoption of vl , ensuring better patient outcomes , and enabling vulnerable patient groups and those in rural areas to be reached more effectively . however , careful consideration will need to be given to ensuring quality control and performing cost - effectiveness analysis of true poc testing , and operational research to assess which technologies could be adapted to which contexts is needed [ 2224 ] . if countries are to better optimize the mix of poc and laboratory - based tools , they will need to increase the functioning of their referral networks , including sample and results delivery ( to clinics and patients ) through preexisting referral networks and other electronic and mobile solutions [ 25 , 26 ] . this will not only decrease the cost per result through reduced transport costs and reductions in missing data , but also improve service delivery by providing rapid results and better linkage to care . the global fund 's procurement strategy for vl and eid now places a welcome emphasis on supporting significant investments in laboratory systems and sample transport networks and people ( http://www.theglobalfund.org/en/procurement/viral-load-early-infant-diagnostics/ ) . although prices have continued to come down for vl testing , with the entry of new products and manufacturers to the market , there is still considerable room for price decreases and numerous price - decreasing mechanisms that could and should be adopted . the market is still dominated by a handful of suppliers , and incentives and/or procurement flexibility will be needed to ensure new manufacturers can enter the market to drive prices down . there remains a considerable gap between the real costs of manufacture and the actual prices paid by countries for reagents and consumables as well as shipment , customs charges , tax , service , and maintenance , etc demonstrating the potential for further price decreases . one study found that the costs associated with intellectual property are significant , with royalty payments for some vl technologies accounting for a considerable portion ( 19%63% ) of the total manufacturing costs . intellectual property should , ideally , be licensed at low or no cost when the final products are sold to lmics , and any cost saving should be passed on to purchasers . global health actors will need to better support strategies , such as pooling patents from third parties , royalty - free patents , or reasonable royalties , to ensure affordability . an analysis from the global fund 's price and quality reporting tool found that vl reagent costs alone varied from $ 13.13 to $ 43.34 between countries . publicly available data put the costs for reagents and consumables negotiated by the clinton health access initiative in kenya at only us$10.50 per test , suggesting considerable flexibility on price and the need for greater transparency between governments and other purchasers to ensure that more programs are able to access the best prices . in high - volume situations , donors and affected governments should encourage competition between contractors and negotiate for lower prices ( including for maintenance ) and better services . in brazil , kenya , and south africa , for example , as a result of competition at the tendering stage and negotiation , the selling price per laboratory - based test has now dropped to around us$10.00 , which includes the cost of instrument rental and service and maintenance ( although not human resources or transport services ) . the south african tender also included provisions to create a global price ceiling , which allows 83 countries to access one company 's reagents for < $ 9.40 per test ( http://www.theglobalfund.org/en/procurement/viral-load-early-infant-diagnostics/ ) . as an extension of this price ceiling , roche molecular diagnostics offers ex - works pricing for both vl and eid at $ 9.40 per test , including regents and proprietary consumables , for lmics eligible under their global access programme ( http://molecular.roche.com/globalaccessprogram/documents/gap_country_list_15july2015.pdf ) . furthermore , moving to pooled procurement and to instrument rental options rather than purchasing expensive equipment that does not allow for flexibility in platform choice in the future are options that ensure better prices and service agreements for lmics . for example , the global fund , the us president 's emergency fund for aids relief , and high - volume countries such as south africa currently have the opportunity to pool volumes , which will increase purchasing power , allow for more attractive split tenders , and result in lower prices . future tenders should include the option for manufacturers of polyvalent platforms to submit bundled pricing discounts across disease testing platforms ( tuberculosis , hiv , hepatitis b virus , and hepatitis c virus , etc ) , as abbott molecular has done in the recent global fund tender , and the tender criteria should be transparent . last , negotiations with manufacturers for large volumes , optimizing throughput ( efficiency ) of each instrument , and considering the use of open , multimanufacturer platforms to further stimulate choice and competition , should further reduce the cost per test . it will be useful for manufacturers to have 18-month forecasting estimates to be able to better plan manufacturing need and improve efficiency and , therefore , more easily offer price reductions . driving down the costs of reagents and consumables offers the greatest potential for overall vl cost reductions , contributing to > 60% of vl testing costs according to a comprehensive costing survey across sites in 6 countries ( kenya , thailand , lesotho , malawi , swaziland , and zimbabwe ) . these fully loaded costs ranged from $ 21.56 to $ 44.07 and included costs attributed to human resources , sample collection , reagents and consumables , equipment , laboratory , quality control , results delivery , and costs due to machine failures . what is now clear is that diagnosis and monitoring is key to early and accurate disease detection and ensuring treatment efficiency , and thus our ability to tackle hiv / aids in lmics ( http://www.unaids.org/en/resources/documents/2014/90-90-90 ) . in our view , governments should be looking at allocating up to 15% of total hiv program budget on facility - level costs that should go toward laboratory testing , including training and support for laboratory personnel . of key importance is the need for governments and donors to prioritize the strengthening of health and laboratory systems globally [ 10 , 15 ] . maintaining a strong workforce , training them in delivering new tests and task shifting policies that allow nonphysicians , including nurses and trained lay workers to administer testing and treatment is a crucial first step . msf 's survey highlighted that all 5 countries reported insufficient personnel availability , with overburdened and inadequate numbers of healthcare professionals in front - line facilities , as well as insufficient training and a lack of up - to - date knowledge . investments in vl sample transport , laboratory tools , reporting tools , databases , and human resources can be leveraged to benefit other diseases too , accelerating diagnostic access overall and health systems strengthening [ 7 , 28 ] . as countries consider which platforms to select , polyvalency the capacity to run assays for other diseases on the same platform should be considered . this approach reduces overall large upfront costs , because a different machine does not need to be purchased or rented for each different disease , and facilitates standardized human resource training , service and maintenance , and procurement . program managers should consider a leasing or reagent rental option , to allow flexibility to adopt newer and more efficient technologies as and when they come to the market and to ensure an all - inclusive contract . improving sample transport for example , by strengthening transport networks to ensure the prompt delivery of results to people is also crucial . the use of dbss has greatly simplified sample transport to the laboratory from remote sites , and new advances in dbs technology will undoubtedly increase accuracy [ 7 , 29 ] . despite the fact that more data are needed to inform next steps to scaling up dbs at the present time , what is clear is that in many current settings dbs is the only option for ensuring scale - up of vl testing . use of pooled samples , whereby dbs samples ( either via a fingerprick or phlebotomy ) from multiple people ( eg , minipools of 5 samples ) are mixed together and 1 test is conducted on the pooled sample , has been shown in several studies to reduce costs related to vl monitoring , particularly where a high percentage of patients ' vl is suppressed . in malawi , using a cost of us$30 per vl , dbs pooling reduced the number of tests performed by 30% , saving $ 207 000 per year . as prices for vl testing continue to drop , the cost - effectiveness of pooling samples may decline . pooling is only useful if it saves money , so countries will need to make their own decisions according to test volume and failure rate . where it is not feasible to fully introduce routine vl testing , a phased implementation approach could be a positive first step in facilitating logistical and technical laboratory capacity , and could be established before widespread scale - up [ 6 , 8 ] . one possible approach is to prioritize particular higher - risk patients for example , to use vl just to confirm clinical or immunological treatment failure before switching art or to perform routine vl specifically for children and adolescents , or pregnant and breastfeeding women . although these approaches represent suboptimal use of vl testing , they allow phasing in of vl testing while capacity is built for universal access to routine monitoring . last , programs need to consider an education and counseling component , because vl testing can function as a motivator for adherence and informed patients can further create a demand for vl testing . recent years have seen increased and welcomed commitment toward improving both the quality and outcomes of art programs , with data showing that it is both medically important and operationally feasible to implement vl testing in lmics . there have been calls to divert funds from cd4 testing to vl testing [ 31 , 32 ] ; treatment initiation for all hiv - infected people has now been recommended by who in its 2015 guidelines , in response to the recent strategic timing of antiretroviral treatment trial results , continuing preferably with routine vl monitoring but with cd4 testing now reserved for measuring immunosuppression to inform the need for prophylaxis and to screen for opportunistic infections . table 1.challenges and solutions in viral load implementationchallengesproposed solutionspoor adherence to current who guidelines on vl testingcountries should incorporate routine vl monitoring into their national guidelines and may need external support to do thisfunding shortfalls for routine vl monitoring scale - updonors and national governments must prioritize vl testing and scale - up in their budgetshigh costs for vl tests , reagents , and consumablesdecrease the cost of products through a variety of mechanisms ( eg , pooled procurement , competition , negotiation , and pricing transparency)weak health and laboratory systems in low- and middle - income countriesstrengthen health and laboratory systems globally , addressing human resource and training issues : 15% of an hiv program budget should be earmarked for laboratory / diagnosticsweak transport systems and networks for delivering resultsstrengthen these systems , using approaches such as e- and m - health solutions , and more convenient sample strategies such as dried blood spotslow levels of staff training and quality assurancelaboratories should work toward becoming accredited , such as via the stepwise laboratory improvement process towards accreditation program , and follow the new who guidelines on ensuring the quality of hiv - related poc testing , and implement quality assurance / quality control and proficiency testinglack of awareness among patients and clinicians as to the benefits of vl testingeducation empowerment work , adopting community - centric strategieslack of civil society mobilization on the issue of vl testing and access to diagnosticsencourage their involvement in advocating for rapid and appropriate scale - up of vl testing , adopting lessons learned from antiretroviral therapy scale - uppoor awareness among program managers as to the wider benefits of vl scale - up to health - system strengthening and cost reduction program managers should consider polyvalent technologies to enable testing for multiple disease and bundled pricing , and multimanufacturer platforms sourcing products from multiple suppliers to bring costs down through competitionprogram managers should consider a leasing or reagent rental option and bundled pricing across diseases , to allow flexibility to adopt newer and more efficient technologies as and when they come to the market and to ensure all - inclusive services , and bundled pricing across diseases to leverage price gains made for hivlimited local and national guidance on supporting patients with a low vlintroduce community - centric models of care that allow people to receive treatment in the community and not visit the clinic more than once a year.centralized or decentralized?when deciding between a centralized laboratory - based approach and a decentralized or near - poc approach , take several factors into account , including patient volume and characteristics , cost per test , throughput per site , anticipated levels of instrument usage , human resource skill level , and task - shifting policiescould a phased implementation approach be an option?yes , for example using vl testing on selected higher - risk populations . this is considered to represent suboptimal use of vl testing but could be considered to kick - start scale - upabbreviations : hiv , human immunodeficiency virus ; poc , point - of - care ; vl , viral load ; who , world health organization . handbook for improving the quality of hiv - related point - of - care testing . ensuring the reliability and accuracy of test results . in press . challenges and solutions in viral load implementation program managers should consider polyvalent technologies to enable testing for multiple disease and bundled pricing , and multimanufacturer platforms sourcing products from multiple suppliers to bring costs down through competition program managers should consider a leasing or reagent rental option and bundled pricing across diseases , to allow flexibility to adopt newer and more efficient technologies as and when they come to the market and to ensure all - inclusive services , and bundled pricing across diseases to leverage price gains made for hiv abbreviations : hiv , human immunodeficiency virus ; poc , point - of - care ; vl , viral load ; who , world health organization . handbook for improving the quality of hiv - related point - of - care testing . ensuring the reliability and accuracy of test results . in press . what is now becoming clear is that vl testing has numerous cost benefits for hiv programs , facilitating a reduced number of clinical contacts and opportunities for further task shifting , as well as reducing the cost of drugs by preserving first - line art and reducing transmission [ 34 , 35 ] . vl monitoring integrated with intensive adherence support programs has been shown to be successful in increasing adherence to art , thus preventing a treatment switch [ 3 , 36 ] . additional research is needed to optimize the package of adherence support and visit spacing linked with routine vl monitoring . training for clinicians , to be better able to provide optimal care , as well as people living with hiv / aids to know how to best monitor their health , is increasingly becoming important . since civil society have advocated for many of the most important tools and policies that have improved the hiv response globally , their voice will now be more important than ever in demonstrating demand and ensuring the rapid and appropriate scale - up of vl testing .

in the era of assisted reproduction , we have potential to achieve pregnancy for women at any age . irrespective of menstrual functioning of the women our ability to make this possible with advent of donor embryo transfer have given us a sense of pride and potential to have near control on reproduction biology , but it raises the question of how ethically are we correct . ignoring the unforeseen complications associated with conception at an elder age can lead to increased morbidity for the expectant mother . such patients are at high risk for developing multiple complications during pregnancy , and we came across with one such case which needed a multidisciplinary approach in managing postmenopausal pregnancy . a 65-year - old postmenopausal primigravida who conceived after donor embryo transfer was referred to our center at 29 weeks of pregnancy with antepartum hemorrhage . her previous antenatal monitoring was at the private hospital where she had undergone in vitro fertilization ( embryo donation ) . at the time of admission , her blood pressure was 160/108 mmhg for which she received intravenous labetalol . cardiovascular system examination revealed a grade 3 diastolic murmur , but there were no signs of congestive heart failure . obstetric examination showed uterine height corresponding to the period of gestation with a transverse lie ; uterus was relaxed and regular fetal heart rate . on local examination she was started on oral methyldopa 250 mg thrice a day and labetalol 100 mg thrice a day . she was kept on conservative management and was evaluated further for heart disease and diabetes . echocardiography showed trace tricuspid regurgitation with moderate pulmonary artery hypertension with normal left ventricular function . she was also diagnosed as gestational diabetes mellitus controlled on diabetic diet and insulin therapy . she also received steroid cover for fetal lung maturity . at 32 weeks period of gestation by date of embryo transfer , she had another bout of bleeding ; therefore , emergency lower segment cesarean section was performed . a male child with birth weight 1650 most of the placenta was adherent with no plane of cleavage and hence cesarean hysterectomy was performed . postoperatively , the patient was monitored in the intensive care unit ( icu ) for 24 h. baby was shifted to neonatal icu for observation . in icu , her systolic blood pressure ranged 190220 mm hg and diastolic blood pressure ranged 110130 mmhg . on the 2 postoperative day , she was started on tablet amlodipine 10 mg per orally once daily . this case is one more example of maintenance of uterine receptivity to embryo implantation and development well beyond the natural menopause if adequate hormone replacement is given . used a uniform preparatory cycle of hormone replacement and found no differences in the histologic , ultrasonographic , and tissue receptor response of women 5060 years of age and younger recipients 2540 years of age . one of the largest series of delivered pregnancies in women over the age of 50 years showed outcomes were good although gestational hypertension was seen at a much higher incidence than expected and same was seen in our patient . it also concluded that though pregnancy rates and risk of multiple gestation after oocyte donation is similar as in younger population but higher obstetric surveillance and care is required due to more antenatal complications in patients over the 50 years . postmenopausal pregnancy ( pmp ) though helps the couple to fulfill their wish of having their own child , raises several issues such as like ethics , psychological , and health aspects . oocyte donation has been used over a decade to establish pregnancies in functionally agonadal women . landau et al . who examined the psychosocial , health , and ethical aspects of pmp concluded that it increases human suffering rather than decreasing it . older women are more likely to suffer from comorbidities such as hypertension , diabetes , and heart disease and all these three were present in this case . in addition , they are at a risk of obstetric complications such as morbidly adherent placenta and intrauterine growth restriction . although pregnancy is possible in postmenopausal woman with hormone support , the incidence of complications remains very high . it raises a need for developing well - laid guidelines for performing ivf in elder age group women . furthermore , counseling the couple regarding pros and cons of pmp will help them weigh risks against benefits for the same .

qat leaves contain more than 40 alkaloids , glycosides , tannis , terpenoids and others . cathine and cathinone are the most important alkaloid constituents of qat that have amphetamine like effect . therefore , people chew qat to get psycho - stimulation effect in the form of euphoria and excitement due to its cathin and cathinone contents . millions of men and women practice this habit in yemen society in addition to ethiopia , somalia and other countries of east africa . qat habit is known in the arabic countries as takhzeen al - qat as it is more than chewing ( chewing and storing qat in the vestibule for few hours ) and known socially as qat sessions . tachycardia , palpitation , hypertension , gastritis , constipation , bronchodilation and relaxation of bladder wall are common examples of qat effects on the human body [ 1 , 46 ] . the clinico - pathological effects of qat habit on the oral mucosa are well documented [ 3 , 7 , 8 ] . these effects include different grades of keratotic white lesions , keratinization of non - keratinized oral mucosa and epithelial dysplasia . recent genetic study showed that qat consumption , especially when accompanied by alcohol and tobacco consumption might be a potential cause of oral malignancy . this finding was supported by another study , which considers the qat habit as a probable contributing etiological factor of scc , however , this is not the case in other studies [ 3 , 7 , 11 ] . despite millions in yemenis community chew qat , the proportion of the population with severe periodontal disease in yemen was reported to be significantly lower than in countries with high level of dental care [ 1215 ] . another clinical study showed that qat consumption may affect the periodontal tissue especially among younger probands . however , they did not study clearly the role of different variables of qat , mainly , the duration and frequency of chewing . the controversies regarding the association of periodontal diseases and the public habit of takhzeen alqat in yemen community motivated us to conduct this study . this cross - sectional study was done on 2500 yemenis randomly reported to the faculty of dentistry , sanaa university , mean age 27.01 years ( sd=13.4 ) , of them 1818 ( 72.7% ) male and 682 ( 27.3% ) female . a special questionnaire was designed for this study and revised to include maximum information of the exposure under study qat chewing habit and its effects . the questionnaire was filled for each case with a full intra - oral clinical examination using the normal examination tools , periodontal probe and dental unit light . in addition to the evaluation of the periodontal status , the questionnaire and clinical examination included evaluation of the buccal mucosa and associated white lesions which had been published elsewhere . questionnaire included the following : personal data : case number , age , sex and occupation.habits information : a chewer ( 1 ) or a non - chewer ( 0 ) , if chewer , how many years he has been a chewer ( 140 ) , and frequency ( number of qat chewing sessions per week ) ( 17).any other habit that may affect the oral mucosa such as : smoking : a smoker ( 14 ) or a non - smoker ( 0 ) , if he is a smoker it s frequency has been recorded as : ( 1 ) less than 10 cigarettes daily . ( 2 ) more than 10 and less than 20 cigarettes daily . ( 3 ) more than 20 cigarettes daily . and ( 4 ) ex - smoker ( a person who left smoking for more than 1 year ) . madaa ( water pipe ) : a smoker ( 1 ) or a non - smoker ( 0).shamma ( smokeless tobacco ) or what so called in some regions tombak : a user ( 1 ) or a non - user ( 0).any other habits such as tumbol or betel nut chewing : a user ( 1 ) or not a user ( 0 ) . personal data : case number , age , sex and occupation . habit s information : a chewer ( 1 ) or a non - chewer ( 0 ) , if chewer , how many years he has been a chewer ( 140 ) , and frequency ( number of qat chewing sessions per week ) ( 17 ) . any other habit that may affect the oral mucosa such as : smoking : a smoker ( 14 ) or a non - smoker ( 0 ) , if he is a smoker it s frequency has been recorded as : ( 1 ) less than 10 cigarettes daily . ( 4 ) ex - smoker ( a person who left smoking for more than 1 year ) . madaa ( water pipe ) : a smoker ( 1 ) or a non - smoker ( 0).shamma ( smokeless tobacco ) or what so called in some regions tombak : a user ( 1 ) or a non - user ( 0).any other habits such as tumbol or betel nut chewing : a user ( 1 ) or not a user ( 0 ) . smoking : a smoker ( 14 ) or a non - smoker ( 0 ) , if he is a smoker it s frequency has been recorded as : ( 1 ) less than 10 cigarettes daily . ( 4 ) ex - smoker ( a person who left smoking for more than 1 year ) . madaa ( water pipe ) : a smoker ( 1 ) or a non - smoker ( 0 ) . shamma ( smokeless tobacco ) or what so called in some regions tombak : a user ( 1 ) or a non - user ( 0 ) . any other habits such as tumbol or betel nut chewing : a user ( 1 ) or not a user ( 0 ) . periodontal probing was done in chewers in the area of takhzeen al - qat only ( s1 , s3 , s4 , and s6 ) and compared with the same sextants in non users of qat ( posterior first and second molars ) using the normal periodontal probe . two measurements were taken : the distance in millimeters from the free gingival margin to the base of the pocket ( pocketing ) , and from the cervico - enamel junction to the free gingival margin ( gum recession ) . the periodontal probe was gently inserted into the gingival sulcus of each tooth until light resistance is met and then it was moved around the tooth s circumference . the greatest probe depth in the mentioned area of the mouth was recorded . the following variables of the periodontal status were scored : gum bleeding : ( 0 ) no bleeding , ( 1 ) with bleeding . ( 2 ) excluded sextant.pocketing : ( 0 ) less than 3 mm . ( 1 ) from 3 to 5 mm . ( 2 ) 6 mm or more . ( 3 ) any pocketing with gum recession . ( 4 ) excluded sextant . ( 1 ) from 3 to 5 mm . ( 2 ) 6 mm or more . ( 3 ) any pocketing with gum recession . ( 4 ) excluded sextant . all collected data has been fed into a specialized computer program ( spss ) for data analyses , and by using chi test , reliable results were only documented when p0.05 . questionnaire included the following : personal data : case number , age , sex and occupation.habits information : a chewer ( 1 ) or a non - chewer ( 0 ) , if chewer , how many years he has been a chewer ( 140 ) , and frequency ( number of qat chewing sessions per week ) ( 17).any other habit that may affect the oral mucosa such as : smoking : a smoker ( 14 ) or a non - smoker ( 0 ) , if he is a smoker it s frequency has been recorded as : ( 1 ) less than 10 cigarettes daily . ( 2 ) more than 10 and less than 20 cigarettes daily . ( 3 ) more than 20 cigarettes daily . and ( 4 ) ex - smoker ( a person who left smoking for more than 1 year ) . madaa ( water pipe ) : a smoker ( 1 ) or a non - smoker ( 0).shamma ( smokeless tobacco ) or what so called in some regions tombak : a user ( 1 ) or a non - user ( 0).any other habits such as tumbol or betel nut chewing : a user ( 1 ) or not a user ( 0 ) . personal data : case number , age , sex and occupation . habit s information : a chewer ( 1 ) or a non - chewer ( 0 ) , if chewer , how many years he has been a chewer ( 140 ) , and frequency ( number of qat chewing sessions per week ) ( 17 ) . any other habit that may affect the oral mucosa such as : smoking : a smoker ( 14 ) or a non - smoker ( 0 ) , if he is a smoker it s frequency has been recorded as : ( 1 ) less than 10 cigarettes daily . ( 2 ) more than 10 and less than 20 cigarettes daily . ( 3 ) more than 20 cigarettes daily . and ( 4 ) ex - smoker ( a person who left smoking for more than 1 year ) . madaa ( water pipe ) : a smoker ( 1 ) or a non - smoker ( 0).shamma ( smokeless tobacco ) or what so called in some regions tombak : a user ( 1 ) or a non - user ( 0).any other habits such as tumbol or betel nut chewing : a user ( 1 ) or not a user ( 0 ) . smoking : a smoker ( 14 ) or a non - smoker ( 0 ) , if he is a smoker it s frequency has been recorded as : ( 1 ) less than 10 cigarettes daily . ( 2 ) more than 10 and less than 20 cigarettes daily . ( 3 ) more than 20 cigarettes daily . and ( 4 ) ex - smoker ( a person who left smoking for more than 1 year ) . madaa ( water pipe ) : a smoker ( 1 ) or a non - smoker ( 0 ) . shamma ( smokeless tobacco ) or what so called in some regions tombak : a user ( 1 ) or a non - user ( 0 ) . any other habits such as tumbol or betel nut chewing : a user ( 1 ) or not a user ( 0 ) . periodontal probing was done in chewers in the area of takhzeen al - qat only ( s1 , s3 , s4 , and s6 ) and compared with the same sextants in non users of qat ( posterior first and second molars ) using the normal periodontal probe . two measurements were taken : the distance in millimeters from the free gingival margin to the base of the pocket ( pocketing ) , and from the cervico - enamel junction to the free gingival margin ( gum recession ) . the periodontal probe was gently inserted into the gingival sulcus of each tooth until light resistance is met and then it was moved around the tooth s circumference . the greatest probe depth in the mentioned area of the mouth was recorded . the following variables of the periodontal status were scored : gum bleeding : ( 0 ) no bleeding , ( 1 ) with bleeding . ( 2 ) excluded sextant.pocketing : ( 0 ) less than 3 mm . ( 1 ) from 3 to 5 mm . ( 2 ) 6 mm or more . ( 3 ) any pocketing with gum recession . ( 4 ) excluded sextant . ( 2 ) 6 mm or more . ( 3 ) any pocketing with gum recession . ( 4 ) excluded sextant . all collected data has been fed into a specialized computer program ( spss ) for data analyses , and by using chi test , reliable results were only documented when p0.05 . this study was done on 2500 yemeni , of them 1528 ( 61 % ) were qat chewers and 972 ( 39 % ) were non - chewers . in our sample , 1330 ( 73.2 % ) of males were qat chewers and 198 ( 29 % ) of females were qat chewers . out of 972 non - chewers 116(12% ) had periodontal pocketing of different depths , 473 ( 48.7% ) cases had gingival bleeding and 18 ( 1.9% ) had gum recession . on the other hand , out of 1528 chewers , 468 ( 31.8% ) had periodontal pockets , 1214 ( 79.5% ) with gum bleeding and 98 ( 6.4% ) had gum recession . regarding the periodontal status and frequency of chewing , it was found that 536 yemenis chew qat only once per week , of them 100 ( 18.6% ) had periodontal pockets . 719 were found to be daily qat chewers with 269 ( 37.9% ) of them having periodontal pockets . with respect to gum bleeding and frequency of chewing , there were no differences between persons who chew qat one day per week and others who chew qat daily . with respect to the relationship between duration of chewing and periodontal status , it was found that 53 persons were qat chewers for less than 1 year ; 4 of them ( 7.5% ) had periodontal pockets . 371 were qat chewers for more than 10 years , out of which 258 ( 69.5% ) had periodontal pocketing . though qat is well documented to cause many clinico - pathological changes in the oral mucosa [ 3 , 7 , 8 ] , yemeni population who chew qat commonly were showed to have low prevalence rate of periodontitis . a clinical study reported that the proportion of the population with severe periodontal disease in yemen is significantly lower than in countries with high level of dental care . as individuals chew qat only at the posterior teeth ( s1 , s3 , s4 , and s6 ) , the first and second molars only were examined while anterior sextants ( s2 and s5 ) were excluded from the study . results of this study showed that qat is very harmful to the periodontal tissue as 79.5% , 31.8% and 6.4 of chewers had gum bleeding , periodontal pockets and gum recession respectively , while 48.7% , 12% and 1.9% of non - chewers had gum bleeding , periodontal pockets and gum recession respectively ( p<0.05 ) . this is in contrast to another study which could not find a causal relationship between the use of qat and periodontal disease . differences between results in the two studies might be related to the differences in the sample size and the geographical area where sample was selected [ as this study was done in sanaa only while the other study was done in different cities in yemen ] . moreover , it did not focus on the role of different variables of qat ; mainly , the duration and frequency of chewing . the mechanism by which qat may affect the periodontal tissues might be related to the trauma and/or the microbiological role . trauma induced by the habit of takhzeen al - qat was suggested to cause pathological changes in the oral mucosa as white lesions . these white lesions usually appear in the area of takhzeen al - qat including the oral mucosa or the gingiva . such trauma induced by qat fibers during the habit session ( 35 hours ) or by remnants of qat fibers between teeth after the qat session may play an important role in the development of periodontal diseases . qat constituents may affect on the normal oral flora , which might also be a possible cause for these periodontal diseases . this could be supported by the recent study which showed that takhzeen al - qat induces a microbial profile that is not incompatible with gingival health . epidemiologically , if we suggest a relationship between a causal factor and a disease , the risk of developing this disease will increase when the exposure to the risk factor increases . this was very clear in this study as the risk of developing periodontal diseases increased when the exposure to the risk factor ( duration and frequency of takhzeen al - qat ) increased . in consequence with this fact , results of this study showed that 37.9% of persons who chew qat daily had periodontal pockets while 18.6% of persons who chew once per week had similar changes . similarly , it was found that 69.5% of persons who chewed qat for more than 10 years had periodontal pockets while only 7.5% of persons who chew qat for less than 1 year had similar lesions ( p<0.05 ) . gum recession appeared in 10.4% of persons who chew qat daily and in 2.2% of persons who chew qat once per week . similarly , it appeared in 4.3% of persons who chewed qat for more than 10 years and in 1.3% of persons who chewed qat for less than a year . all these differences support the epidemiological hypothesis which states that increased exposure to qat habit will increase the risk of developing periodontal pockets and gum recession . in conclusion , this study can propose the habit of takhzeen al qat as an etiological factor for periodontal diseases .

hugonnet and colleagues present an interesting article on nurse staffing and ventilator - associated pneumonia ( vap ) . although this study joins a number of other studies on nurse staffing and adverse outcomes of hospitalized patients , i feel compelled to address several important limitations of this study . what i find disappointing is the fact that these authors describe an observational study in which i expect they had an opportunity to add something substantial to the body of literature on nurse staffing and adverse health care related outcomes , but failed to do so . there is sufficient evidence in the literature to suggest that nurse staffing is significantly associated with health care associated infections [ 2 - 4 ] , but we lack data on the process of nursing care that may very well inform us as to why this staffing association exists . the authors do state that some of the process of care measures were not consistently recorded , but they do not state that all of those measures were missing . because these data on the process of care are of such importance , i would hope that the authors considered a method of imputation before making the decision to eliminate these data from the analysis . they control for central venous , peripheral , and urinary catheters , but it would have been of great value to include data on nurse process of care measures , such as the presence or absence of mouth care , which is a potential risk factor for vap . in addition , i am surprised that the authors did not include hand hygiene as a risk factor of interest , because there is a well established link between hand hygiene and health care associated infections , and one that the authors have worked with extensively . the authors painstakingly constructed a comprehensive risk adjustment model that includes , but is not limited , to the charlson comorbidity index , the acute physiology and chronic health evaluation ii score , and the projet de recherch en nursing acuity score . i am concerned , however , that these measures overlap and , although not mentioned , i hope that the authors verified that there were no issues with collinearity . the cox hazards model is an appropriate choice when measuring time to vap , but i have a few concerns surrounding the method of censoring and construction of the variables that are time dependent . i can understand the exposure period for the nurse staffing variable , but i think it best to construct all other of the time - dependent variables as days from admission to censoring . as for censoring , i also do not agree with censoring 5 days post - extubation . the authors ' choice of censoring prohibits taking into account the patients who might well have experienced respiratory compromise and required re - intubation . because the extubated patients were censored ( removed from the analytic model ) on day 5 after extubation , these patients are no longer included in the sample for analytic purposes , even though they are still presumably at risk for vap . what i found most troublesome with this analysis is how the authors computed what they refer to as the risk factor of interest , namely nurse staffing . they refer in the text to the nurse staffing per shift , and in fact they provide the median nurse - to - patient ratio for the morning , evening , and night shifts as 0.8 , 0.6 , and 0.6 , respectively . however , in the final hazard models nurse staffing is computed as the total number of nurses working in a 24-hour day divided by the patient census . such a computation inflates the nurse - to - patient ratio , as indicated by the fact the median daily ratio ranged from 1.4 to 5.3 nurses per patient . nurse staffing has been computed differently in a number of studies in the literature , such as full - time equivalent registered nurses per adjusted inpatient day , registered nurse hours per adjusted inpatient day , and nurse - to - patient ratio ; although these computations differ , the final recommendations make some sense from an administrative point of view . the estimation by the authors that hospitals maintaining a nurse - to - patient ratio above 2.2 could decrease the risk of health care associated infections is based on findings that are potentially biased and unrealistic . even though hugonnet and colleagues provide what i consider to be suboptimal estimates of nurse - to - patient ratio , i applaud their attempt to forge along the causal pathway that links nurse staffing to health care associated infections in an attempt to improve the quality of patient care . we thank cimiotti for her detailed commentary on our study and focus on the few relevant criticisms she makes in our response . although knowledge in this field is still partial and further research is required , our study adds to the increasing evidence in the literature that adequate staffing is a prerequisite for patient safety . it provides additional data on the epidemiology of vap ; few studies have investigated the association between workload and pneumonia , and none have specifically focused on late - onset vap . the optimal method with which to estimate how much time and care each patient received in order to derive some sort of an ' offer / demand ' ratio would be to measure it individually , but this is unrealistic . computing a workload measure per shift or over 24 hours neither is there any fundamental difference between measuring nurse - to - patient ratio , full - time equivalent nurses , or number of nurse hours per patient . these details should not blur what is by far the main problem ; these measures are all of an ecological nature and this is seldom acknowledged . because we investigated only vap , the analysis of time or time at risk can not start before initiation of mechanical ventilation , and precisely how long a patient remains at risk after extubation is unknown . we agree that 5 days is an arbitrary cut - off value , but it seems very reasonable to assume that a pneumonia developing 7 days after extubation is unrelated to mechanical ventilation , as long as there is no intervening re - intubation . of note , a patient who was extubated and re - intubated 3 days later was still in the at - risk period and included in the analysis . we agree that the process of care is an important issue , but lies in the causal pathway between workload and infection . however , the priority is surely not to demonstrate that busy health care workers do not fully comply with infection control recommendations , but rather to improve the process of care , define adequate staffing levels , and refine statistical and mathematical techniques in risk factor analysis .

the helicobacter pylori ( h. pylori ) bacterium is responsible for 5.5% of all infection - associated cancers and is the major cause of gastric cancer in consequence of chronic inflammation . persistent gastric mucosa inflammation results in chronic gastritis and progresses through a multistep process to gastric atrophy , intestinal metaplasia , dysplasia , and finally carcinoma . the clinical consequences of h. pylori infection are determined by bacteria virulence genes as well as by host genetic factors such as immune response genes , besides environmental factors [ 35 ] . among the bacterial products , the caga ( cytotoxin - associated gene a ) and vaca ( vacuolating cytotoxin ) proteins are the major virulence factors related to the severity of gastric lesions and cell responses [ 6 , 7 ] . the gastric epithelium cells provide the first point of contact for h. pylori adhesion through interaction with toll - like receptors ( tlrs ) , responding to the infection by activating various signaling pathways . tlrs are key regulators of both innate and adaptive immune responses , recognizing several microbial products , such as lipoproteins , peptidoglycans , and lipopolysaccharides ( lps ) . the lps of h. pylori is recognized mainly not only by tlr4 , but also by tlr2 , which recognizes other forms that are structurally different from those recognized by tlr4 . both tlr2 and tlr4 are activated , after the bacteria recognition , in cooperation with the adapter molecule myd88 , triggering the mitogen - activating protein kinase ( mapk ) signaling pathway . at this point , there is a subsequent activation of the transcription factor nf-b , which leads to the rapid expression of inducible nitric oxide synthase ( inos ) and proinflammatory cytokines , chemokines and their receptors , and interleukins [ 12 , 13 ] . when these factors are stimulated , they initiate a marked inflammatory response of the mucosa , characterized as chronically active gastritis , and may acquire oncogenic potential [ 14 , 15 ] . so far , the strategy for prevention of h. pylori - associated gastric cancer has been the eradication of these bacteria , regarded as a first - line therapy to reverse the preneoplastic lesions and prevent malignant progression . however , treatment is not adopted for asymptomatic carriers in developing countries , due to its high cost . h. pylori is susceptible to most antibiotics , although resistance has been common , and triple or quadruple therapy consisting of two antibiotics , a proton pump inhibitor , and bismuth has lately been used to eradicate the bacteria . studies to evaluate changes in expression levels of genes involved in the recognition of the bacteria and the immune response of the host in patients infected by h. pylori are scarce , both before and after eradication treatment . moreover , there are no reports about the expression of tlr2 and tlr4 in gastric lesions before and after bacterial clearance . therefore , the main goal of the present study was to evaluate , for the first time , the mrna and protein expression levels of tlr2 and tlr4 in h. pylori - infected chronic gastritis patients and the occurrence of changes in the expression levels of these receptors after successful h. pylori eradication therapy . at first , 59 patients scheduled for upper endoscopy with positive histological and molecular diagnosis for h. pylori and not yet submitted to eradication therapy were enrolled prospectively between may 2010 and december 2012 from the gastro - hepatology outpatient clinic at the base hospital and the joo paulo ii hospital , both at so jos do rio preto , sp , brazil . from each patient , gastric biopsies of the antrum region were collected for histological analyses and molecular and immunohistochemical studies . none of the individuals had taken any antibiotics , nonsteroidal anti - inflammatory drugs , or corticosteroids during the two months prior to endoscopy , nor did they take proton pump inhibitors or h2 antagonists in the 15 days preceding the procedure . gastric biopsy specimens were examined histologically by a specialized pathologist for the presence of the bacteria and histopathologically classified as superficial chronic gastritis ( n = 45 ; mean age 44 years ; 19 females and 17 males ) , atrophic gastritis ( n = 8 ; mean age 50 years ; 3 females and 5 males ) , and atrophic gastritis with intestinal metaplasia ( n = 6 ; mean age 50 years ; 4 females and 2 males ) , according to the sydney system , constituting the so - called cg - hp+ group . of the 59 cg - hp+ patients , only 37 ( 63% ) concluded the treatment and were called completed treatment group , and 23/37 ( 62% ) of them had the bacteria eradicated , as evidenced by concordant histological and molecular h. pylori - negative diagnosis . however , 14/37 ( 38% ) remain infected showing histological or molecular h. pylori - positive diagnosis ( table 1 ) . four gastric biopsy specimens presented histologically normal h. pylori - negative gastric mucosa ( normal hp- group ) and were used as control ( mean age 35.6 years ; 3 females and 1 male ) . epidemiological data of patients and controls were collected using a standard interviewer - administered questionnaire , containing questions about smoking habits , alcohol intake , previous or ongoing treatment , use of medications , previous surgeries , and family history of cancer . the cg - hp+ group was submitted to standard triple therapy consisting of amoxicillin ( 1 g ) , clarithromycin ( 500 mg ) , and omeprazole ( 20 mg ) , all given twice daily for seven days . three months after treatment , the individuals underwent another endoscopy for collection of gastric biopsies of the antrum region . immediately after collection , the biopsy specimens were placed into rna later solution ( applied biosystems ) and stored at 20c until nucleic acid extraction . the study protocol was approved by the local research ethics committee ( cep / ibilce / unesp number 030/10 ) , and written informed consent was obtained from all participating individuals . dna / rna extraction from the gastric biopsies was performed according to the protocol accompanying the reagent trizol ( invitrogen ) and the concentrations were determined in a nanodrop nd1000 spectrophotometer ( thermo scientific ) . firstly , multiplex pcr was performed , using 100 ng of dna in a final volume of 25 l containing specific primers for h. pylori genes such as urea and tsaa , besides the constitutive human cyp1a1 gene , according to our protocol which was described in previous study . molecular diagnosis was considered positive when at least one gene ( urea or tsaa ) had been amplified . the h. pylori - positive samples were also subjected to pcr for investigation of polymorphisms in the sm regions of the gene vaca as previously described . primers amplify s1 fragment of 176 bp or s2 fragment of 203 bp , while primers for m alleles amplify m1 fragment of 400 bp or m2 fragment of 475 bp . reverse transcription ( rt ) of total rna was performed using a high capacity cdna archive kit ( applied biosystems ) , in a total volume of 25 l , according to the manufacturer 's protocol . then , qpcr was carried out in a steponeplus real time pcr system 2.2.2 ( applied biosystems ) , using specific taqman probes for target genes tlr2 ( assay i d hs00610101_m1 , applied biosystems ) and tlr4 ( assay i d hs01060206_m1 , applied biosystems ) and two reference genes , actb ( part number : 4352935e , applied biosystems ) and gapdh ( glyceraldehyde 3-phosphate dehydrogenase ) ( part number : 4352934e , applied biosystems ) , used as endogenous controls according to the manufacturer 's instructions . all reactions were performed in triplicate in a final volume of 20 l , using 100 ng/l cdna and a blank to ensure the absence of contamination . relative quantification ( rq ) of tlr2 and tlr4 mrna was obtained according to the model proposed by livak and schmittgen and normalized to the actb and gapdh reference genes and a pool of normal hp- samples . immunohistochemical analysis was performed in 14 samples from the cg - hp+ group before and after bacteria eradication and four samples from the normal hp- group . deparaffinized tissue slides were then submitted to antigen retrieval , using a high - temperature antigen - unmasking technique . the sections were incubated with specific primary antibodies : rabbit polyclonal antibody anti - tlr2 ( 06 - 1119 , 1 : 50 dilution ; millipore ) and mouse monoclonal anti - tlr4 ( 76b357.1 , 1 : 200 dilution ; abcam ) . then the slides were incubated with biotinylated secondary antibody ( picture max polymer detection kit , invitrogen ) for 30 minutes , following the manufacturer 's protocol . immunostaining was done with 3,3-diaminobenzidine tetrahydrochloride ( dab ) containing 0.005% h2o2 , counterstained with hematoxylin . placenta mucosa and appendix tissue were used , respectively , as positive controls for the tlr2 and tlr4 proteins . the immunostaining was evaluated in the cytoplasm by densitometric analysis with an arbitrary scale going from 0 to 255 , performed with axio vision software under a zeiss - axioskop ii light microscope . sixty equally distributed points were scored in each one of the regions , and the results were expressed as mean se . the distribution of continuous data was evaluated using the d'agostino and pearson omnibus normality test or shapiro - wilk normality test . data are presented as median and range , as mean standard deviation ( sd ) , or as frequencies , according to the data distribution . student 's t - test for paired and unpaired data or correspondent nonparametric tests , such as the mann - whitney test and the wilcoxon signed rank test , were used for comparisons between groups . to evaluate the association between relative gene expression and risk factors such as age , gender , smoking , drinking , and bacterial virulence genotypes , the correlation between tlr2 and tlr4 mrna expression before and after eradication therapy was analyzed using spearman 's correlation . for protein expression , the means obtained from the densitometry analysis were compared before and after treatment and with the normal hp- group using anova followed by the bonferroni test . the level of significance was set at p 0.05 . table 2 shows the data regarding the relative expression levels of tlr2 and tlr4 mrna of 37 cg - hp+ patients who concluded the treatment ( completed treatment group ) , 23 cg - hp+ patients in which the bacteria were eradicated , allowing paired analysis before and after eradication therapy , and 14 cg - hp+ patients in which the bacteria were noneradicated . the relative expression levels of tlr2 and tlr4 mrna after normalization with the actb and gapdh reference genes and comparison with normal mucosa h. pylori - negative in all groups , either before or after treatment , were increased significantly ( p < 0.05 ) . considering all patients that completed the treatment , no significant change was found after treatment in the relative expression levels of either tlr2 or tlr4 mrna ( tlr2 = 1.55 and tlr4 = 1.64 ) in comparison to the same cases before the treatment ( tlr2 = 1.31 and tlr4 = 1.45 ) . in the group that eradicated the bacteria , heterogeneity of relative expression levels for both tlr2 and tlr4 mrnas can be observed before and after the treatment ( figures 1(a ) and 1(b ) ) . however no significant differences were observed for both genes comparing the expression levels in this group before and after treatment ( p = 0.533 and p = 0.094 for tlr2 and tlr4 , resp . ) furthermore , a positive correlation between the rq values of tlr2 and tlr4 mrna before and after treatment considering only the eradicated patients was found ( before : r = 0.85 , p < 0.0001 ; after : r = 0.55 , p = 0.006 ) . the influence of caga and vaca bacterial genotypes on the gene expression levels , both before and after treatment ( table 3 ) , showed no evidenced significant difference between caga+ and caga genotypes ( p > 0.05 ) for both analyzed genes . we also evaluated the association between relative expression levels of tlr2 and tlr4 mrna and the risk factors such as age , gender , smoking , drinking , and histological type of gastric lesion . none of the factors investigated showed significant differences ( data not shown ) . in normal mucosa , the tlr2 and tlr4 protein expression was weak or absent , mainly in the foveolar epithelium ( figures 2(a ) and 2(b ) ) . nevertheless , the cg - hp+ samples collected before the treatment showed a cytoplasmatic , perinuclear , and focal immunostaining pattern , mostly in the basal area of the foveolar epithelium . a strong expression in the inflammatory cells was also observed ( figures 2(c ) and 2(d ) ) . after the eradication of h. pylori , an immunostaining pattern similar to the one observed before the treatment was found for both tlr2 and tlr4 proteins ( figures 2(e ) and 2(f ) ) . the mean optical densitometry values observed in the normal hp- group for tlr2 and tlr4 were 105.6 2.7 and 101.4 6.5 , respectively . while the cg - hp+ group before treatment presented significantly increased mean values for both tlr2 ( 151.7 6.1 ) and tlr4 ( 132.2 4.7 ) in comparison with the normal hp- group ( p = 0.020 and p = 0.007 , resp . ) . after eradication of the bacteria , both tlr2 and tlr4 proteins showed a slight reduction in their mean optical densitometry values ( 136.1 6.1 and 122.8 5.8 , resp . ) . however , there were no significant differences between these values before and after treatment ( p = 0.064 and p = 0.198 , resp . ) ( figures 2(g ) and 2(h ) ) , confirming the findings regarding the mrna relative expression . in this study we investigated for the first time the occurrence of alterations in the tlr2 and tlr4 mrna and protein expression in h. pylori - infected patients with chronic gastritis , before and after successful bacteria eradication treatment . our results did not reveal significant changes in the relative expression levels of either tlr2 or tlr4 mrna after treatment in eradicated patients , which was confirmed by immunohistochemistry . moreover , the mrna expression of both receptors remained increased after eradication therapy compared to the normal hp- group , showing that the eradication of the bacteria did not normalize the expression of these receptors , at least under the conditions evaluated . additionally , we also observed a positive correlation between the mrna expression values of tlr2 and tlr4 confirming that h. pylori activates both receptors . . lps of gram - negative bacteria are recognized mainly by tlr4 and also tlr2 activating signaling pathways that culminate in an inflammatory response . it is believed that the interaction between bacterial virulence and a genetically susceptible host is associated with more severe chronic inflammation , which may , in the long run , lead to cancer . under normal physiological conditions , the expression of these receptors in the mucosa of the gastrointestinal tract is low due to the action of their antagonists , such as tollip ( toll - interacting protein ) and ppar ( peroxisome proliferator - activated receptor ) that show higher levels in order to prevent inappropriate activation of nonpathogenic antigens [ 2729 ] . in our study , we observed a slightly increased expression of both tlr2 and tlr4 in cg - hp+ patients even after successful h. pylori eradication compared to the noninfected normal mucosa . in children infected with h. pylori , lagunes - servin et al . ( 2013 ) found an increase in the expression of the tlr2 , tlr4 , tlr5 , and tlr9 in the gastric epithelium compared with noninfected children and also an association with pro- and anti - inflammatory cytokines ( il-8 , tnf- , and il-10 ) . these findings confirm that h. pylori has the ability to increase the in vivo expression of tlrs by gastric epithelial cells early during infection in children , starting a chronic and balanced inflammatory process that will continue for decades , and so may contribute to the development of h. pylori - associated diseases later in adulthood . ( 2013 ) observed that , considering the different tlrs of normal h. pylori - negative mucosa , the mrnaof tlr5 was the most expressed , followed by those of tlr2 and tlr4 . furthermore , these authors found tlr2 and tlr4 overexpression in intestinal metaplasia , independent of the h. pylori status , and in the dysplasia / cancer sequence . moreover , upregulation of tlr2 and tlr4 mrna was also observed in h. pylori - associated normal mucosa . these results were confirmed by immunohistochemical analyses , which found an increase in protein expression in h. pylori - infected normal mucosa , further increasing in intestinal metaplasia and dysplasia / carcinoma . these findings suggest that progressive activation of these receptors , initially not only by h. pylori , but also by other pamps ( pathogen - associated molecular patterns ) or damps ( damage - associated molecular patterns ) , at later stages , may play an important role in gastric carcinogenesis and tumor progression . upregulation of tlr4 expression responsiveness to lps and h. pylori in gastric cell lines has also been reported [ 32 , 33 ] . h. pylori infection induced both tlr4 mrna and protein expression in ags cells that were dependent on bacterial load and infection duration . however , the transfection of ags cells with tlr4 sirna followed by the bacterial infection suppressed the expression of this receptor . moreover , lps of h. pylori upregulate tlr4 expression via tlr2 signaling in mkn28 gastric cell lines by the mek1/2-erk1/2 map kinase pathway , leading also to an increase in cell proliferation . conversely , previous studies [ 3537 ] did not observe any relevant role of tlr4 in the cellular recognition of h. pylori in agc cells . these controversial results may be due to differences in the lipid a structures produced by distinct h. pylori strains [ 3840 ] . therefore , the interaction of the bacteria with tlr2 should also be considered , mainly after the first contact with the gastric mucosa , triggering immunologic responses such as induction of il-8 and subsequent activation of nf-b . our study revealed no reduction of the transcript levels of tlr2 and tlr4 or their proteins 3 months after treatment , showing that the successful eradication of h. pylori does not change the expression of these receptors within a short period after the treatment . similarly , garza - gonzlez et al . ( 2008 ) found no quantitative differences in the tlr4 and tlr5 mrna levels either , regardless of the presence or absence of h. pylori in gastric epithelial cells biopsies and ags cells , suggesting that the mrna levels of both receptors may not be influenced by the infection process or at least not at the time points selected for analysis . however , in our study , we observed higher levels of tlr2 and tlr4 mrna and of both proteins in h. pylori - infected mucosa compared to noninfected normal mucosa . it should however be taken into consideration that the posttreatment time elapsed until biopsy collection which may not have been sufficient for mucosal renovation and transcription level normalization . moreover , alterations in mrna expression levels after h. pylori infection eradication therapy have been demonstrated , involving genes associated with cell damage , inflammation , proliferation , apoptosis , and intestinal differentiation [ 43 , 44 ] . this study did not investigate the molecular mechanisms involved in the inflammatory cascade induced by h. pylori infection triggered by tlr4 and tlr2 . therefore , further investigations are needed to clarify the possible involvement of signaling pathway myd88-mapk - nfb as well as the role of ppars ( peroxisome proliferator - activated receptors ) on inhibition of pathway regulating expression of proinflammatory genes and stress kinase pathways [ 31 , 45 , 46 ] , which suppresses inflammation in h. pylori infection . when we compared the expression levels of tlr2 and tlr4 mrna with risk factors and bacterial virulence genotypes , we did not find any association . the studies that assess the effects of caga and vaca virulence factors on the gene and protein expression are controversial . our results evidenced that there were no quantitative differences in the mrna levels of these receptors regardless of caga and vaca status . ( 2008 ) , which demonstrated that the mrna levels of tlr4 and tlr5 in gastric cells both in vivo and in vitro were not influenced by the vaca status , suggesting that this virulence factor may not be involved in the first steps of innate immune - recognition of h. pylori . another study evidenced downregulation of tlrs 2 and 5 and upregulation of tlr9 by h. pylori in human neutrophils regardless of cagpai status and the integrity of t4ss . in conclusion , we report a discrete increase in tlr2 and tlr4 mrna and protein expression in cg - hp+ patients before eradication therapy and the maintaining of this expression pattern even after treatment , suggesting that these receptors remain expressed in the gastric mucosa even after eradication of the bacteria , at least for the period evaluated . therefore , considering the higher risk of malignant progression in patients infected by h. pylori for a long time , further investigations are needed to clarify the changes in the expression of other genes related with the inflammatory cascade induced by bacteria , such as those encoding cytokines and malignant transformation processes as well as the signaling pathways involved .

ce vloaded silica mixed oxide ( ce v ) with a ce : v molar ratio of 1:1 was synthesized by the wet impregnation method.46 , 47 , 48 ce(no3)36 h2o , ( 62 mg for 1 % , 124 mg for 2 % and 250 mg for 4 % ; aldrich99 % ) and voso4xh2o , ( 63 mg for 1 % , 126 mg for 2 % and 252 mg for 4 % ; aldrich97 % ) were dissolved in distilled water ( 50 ml ) , and this was followed by the addition of sio2 ( 2.0 g , aldrich ) . the reaction mixture was continuously stirred for 4 h at rt , and the mixture was further dried in an oven at 110 c overnight , and then calcined in air flow at 500 c for 4 h to obtain the various 1 , 2 , and 4 wt % of mixed oxides of ce / vloaded silica catalysts . a mixture of 5,5dimethylcyclohexane1,3dione 1 ( 1 mmol ) , aromatic aldehyde 2 ( 1 mmol ) , and malononitrile 3 ( 1.1 mmol ) were dissolved in etoh ( 10 ml ) at rt . the 2 % ce v / sio2 catalyst ( 30 mg ) was added to the reaction mixture and continuously stirred for 1 h. the completion of the reaction mixture was monitored by thinlayer chromatography ( tlc ) analysis ( scheme 1 ) . the obtained catalyst was washed with acetone ( 103 ml ) , dried in vacuo at 7080 c for 2 h , and kept aside for use in the next reaction . the filtrate was concentrated to obtain the crude product , which was purified by recrystallization in etoh to afford the target compounds . structures of all the products ( 4 a l ) were established and confirmed on the basis of their spectral data , h nmr , c nmr , n nmr ( ghsqc ) , ftir , and hrms . 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 reorganized for online delivery , but are not copyedited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .

severe acute respiratory syndrome ( sars ) emerged only in late 2002 , but the rapid transmission of the disease worldwide within a few months has led to serious public health concerns . the putative agent of this new disease , identified in march 2003 , is a novel and more pathogenic strain of the commonly occurring coronavirus ( 1,2 ) . cases were initially defined according to syndrome features in the absence of diagnostic tests ( 3 ) . the proportion of persons infected with sars - associated coronavirus ( sars - cov ) whose infection remained subclinical is not known . such information is important , not only to facilitate understanding of the virulence of the virus but , more importantly to determine whether the control measures currently employed are sufficient to halt the spread of the virus . should asymptomatic infection occur in substantial numbers , the virus may continue to spread , despite the isolation of the clinically apparent cases ; however , this would result in the more rapid development of herd immunity in the community . the aim of this study was to determine the seroprevalence of anti sars - cov antibodies in a population of exposed healthcare workers who worked in wards where an outbreak occurred . at the beginning of april 2003 , an outbreak of sars ( diagnosed according to prevailing world health organization guidelines ) occurred in the surgical wards of the singapore general hospital . the source was initially unknown , and all staff and patients in these wards were potentially exposed and were themselves potential sources of the sars virus . to contain the spread , healthcare workers from these wards were either quarantined in their homes for 2 weeks or sequestered with the patients and continued to look after them , adopting full reverse - barrier practices ( 5 ) . subsequent contact tracing pointed to an index case - patient , whose infection led to 38 cases of sars ( in healthcare workers , patients , and visitors ) in these wards and to another 12 cases of sars in the rest of the hospital campus before the outbreak was brought under control 3 weeks later . of the 200 healthcare workers in the surgical wards quarantined or sequestered , sars developed in 17 , and milder symptoms developed in a number of others , which did not qualify for a diagnosis of sars under prevailing who guidelines ( 3 ) . all 200 healthcare workers , comprising doctors , nurses , health attendants , and receptionists in these surgical wards who were quarantined after the initial outbreak , were invited to participate . of these , three had a history of probable sars but had recovered sufficiently to return to work . another group of 12 house officers , who joined the department during the week the study started , were invited to participate as negative controls because they had no prior exposure to known sars patients . participants filled out a questionnaire about symptoms experienced during the preceding weeks and donated a sample of blood by venipuncture ; the serum specimen was stored at 80c until use . immunoglobulin ( ig ) g antibodies to sars - cov were detected by using a dot blot enzyme - linked immunosorbent assay ( elisa ) using a culture- derived , heat - inactivated virus antigen ( e - e ooi , unpub . data ) at a serum dilution of 1:100 . when compared to results of an indirect immunofluorescent assay in a limited study comprising 32 case - patients with clinically diagnosed sars and 977 control serum samples collected before the sars outbreak , sensitivity and specificity were 100% and 99.8% , respectively . samples that tested positive for igg antibodies to sars - cov were further assayed for neutralizing antibodies by using the 50% tissue infective dose ( tcid50 ) method , similar to that previously described ( 6 ) , under biosafety level 3 conditions , in serial twofold dilution , ranging from 1:10 to 1:320 . the virus isolate used in this study , sars - cov 2003va2774 , has been previously sequenced ( 7 ) and was isolated from a patient in whom sars was diagnosed . all assays were carried out in duplicate , and positive serum controls , obtained from a volunteer convalescent - phase sars patient , were included in every run . four samples tested strongly positive by dot blot elisa , although only three of these were positive for neutralizing antibodies with titers of 1:60 , 1:60 , and 1:320 . nine other samples tested weakly positive by the dot blot elisa , although these samples were all negative by neutralization test . analysis of data provided by the questionnaire showed that of the 84 exposed persons in whom sars did not develop , 32 had combinations of various symptoms . this is the first study to examine the seroprevalence of anti sars - cov antibodies in a population with a high likelihood of having been exposed to the virus . the results indicate that all samples positive for neutralizing antibodies were from persons who had symptoms indicative of sars ( table ) . this result strongly validates the current infection control measures to contain the spread of this virus , i.e. , early identification and isolation of case - patients . of the 887 volunteers , 37 had symptoms that were not sufficient to qualify as having probable severe acute respiratory syndrome ( sars ) . upper respiratory : symptoms : runny nose , sore throat , sore mouth or gums . respiratory symptoms : cough , breathlessness , chest pain . gastrointestinal tract symptoms : vomiting , diarrhea , abdominal colic . musculoskeletal symptoms : muscle ache , joint aches . we had chosen to screen the serum specimens at a low dilution to increase their sensitivity , which would then be confirmed by the serum neutralization test . positive specimens could be due to cross - reaction with other coronaviruses ( 7 ) . although negative findings in a small population are difficult to generalize , our results suggest that subclinical infection is not an important feature of sars . we are currently conducting larger population studies to further investigate this finding . in conclusion , in a population of healthcare workers who worked in surgical wards at the time of the outbreak , only those who sought treatment for probable sars had anti sars - cov antibodies , suggesting no subclinical infection . early identification and isolation of cases

tooth eruption has been described as the movement of a tooth from its normal position within the alveolar process towards its functional position in the oral cavity . this process is accompanied by multiple tissue changes , such as resorption and apposition of the alveolar bone and development of the root and periodontium [ 1 , 2 ] . in some instances , anomalies in these physiological phenomena the impaction may be primary , meaning that the tooth has never erupted , or it may be secondary , meaning that the tooth was reimpacted after eruption . local factors contributing to impacted primary teeth include odontomas , ankylosis , congenitally missing permanent teeth , defects in the periodontal membrane , trauma , injuries of the periodontal ligament , precocious eruption of the first permanent molar , defective eruptive force or a combination of these factors . however , in some cases the etiology of tooth impaction is unknown or may have a genetic basis . recent studies have suggested that arrested eruption may relate to local disturbances in the periodontal membrane of the rank - panklopg system . although the incidence of impaction of primary teeth is considered to be rare , unerupted and impacted premolars are a common finding in children . among all primary teeth the mandibular second primary molar has the highest incidence of semi - eruption or ankylosis . unerupted primary molars may cause problems in the dental arch such as space loss , tipping of adjacent teeth , supra eruption of the antagonist and failure of eruption of the permanent teeth underlying the primary teeth [ 3 , 4 , 6 ] . total impaction is considered a very rare phenomenon with very few cases presented in the literature [ 4 , 9 ] . primary failure of eruption is diagnosed when the unerupted tooth is covered by an intact mucosa and radiographs reveal the tooth to be deeply buried in the jaw bone . the purpose of this report was to present a case of an impacted second primary molar positioned inferior to its succedaneous permanent tooth . a 10-year - old boy was referred to the pediatric dental clinic at the dental school of shiraz university of medical sciences , iran with the complaint of absence of one tooth on the left side of his lower jaw . no symptoms of syndromes were evident and his history did not reveal dental trauma or infection . clinical examination showed normal development of the dentition with the exception of a missing mandibular left second primary molar . a panoramic radiograph revealed normal dental development in all quadrants except on the mandibular left side . it showed two developing , unerupted teeth ; namely , the mandibual left second primary molar and its second premolar ( fig 1 ) . based on this information , surgical extraction of the unerupted primary second molar and the radiopaque mass was planned to facilitate the eruption of the second premolar ( fig 2 ) . a passive lingual arch was placed in the lower arch as a space maintainer ( fig 3 ) . the patient was given an appointment every six months for follow - up evaluation of the second premolar retention and any notable findings in the future and for subsequent orthodontic treatment . a follow up panoramic radiograph showed the second premolar eruption path after 13 months ( fig 4 ) . twenty three months after , a coil spring space - regaining appliance was inserted for distal tipping of the mandibular first permanent teeth due to interruption of patient follow up . after 31 months , the teeth had erupted in its normal position ( fig 5 ) . severe infra - occlusion of deciduous molars has been observed relatively infrequently in children affecting only 2.5%8.3% . furthermore , cases of impacted primary molars positioned inferior to the succeeding premolars have been reported as single cases only [ 9,1114 ] . the abnormal position of the second pre - molar is due to early ankylosis of the second primary molar . embryological studies have revealed that the tooth buds of permanent premolars develop in the palatine region of the upper arch and in the lingual region of the lower arch in relation to the enamel organ of the primary teeth . under normal conditions , then it changes its position shifting toward the root of the primary molar . in the present case the permanent second premolar may have developed in a superior and lateral position with respected crown of the impacted primary mandibular second molar , as in the case reported by borsatto et al . kjaer et al estimated that this arrest in eruption occurs before the age of 3 years when the permanent tooth bud in the initial stage is located laterally to the arrested primary molar . other researchers suggested that noneruption of second primary molars could result from abnormal development of the primary molar germ or malposition of the second premolar angle before 1 year of age . sakai et al reported eruption failure of a single mandibular right primary first molar in a 5-year - old boy . it is to be expected that an ankylosed unerupted primary tooth will become more deeply covered by the alveolar bone during growth . although extraction of infraoccluded primary molars should be avoided , because resorption and exfoliation will most often occur within the normal time frame , extraction of an unerupted primary molar is recommended . this is to permit normal eruption of the tooth , to prevent the unerupted tooth from interfering with the development of the premolar and to avoid the risk of cyst formation . treatment options for an impacted premolar include extraction of the primary tooth and patient follow - up without treatment , but with supervision of the eruption process . other treatment strategies are surgical exposure or surgical repositioning with or without orthodontic traction and surgical removal of the erupted premolar .

congenital avf of the popliteal artery is very rare , with few case reports in the literature . an 89-year - old lady was transferred to our hospital with progressive pain in her right calf radiating to her toes over the course of a few days . on presentation the patient had a background history of type 2 diabetes mellitus , hypertension and atrial fibrillation . on examination of the right lower limb there was a blue discoloration of the foot . her right leg appeared to have a greater diameter when compared to the left leg ( fig . no pulse or thrill was palpable at the site of the popliteal artery , nor were the distal pulses palpable . as she had an acute presentation , she did not undergo any pre - operative radiology or vascular investigations . the patient was started therapeutically on a heparin infusion and brought emergently to theatre for a femoral embolectomy . a small embolus was retrieved , however the foley catheter could not pass beyond the level of the popliteal artery . as there was reasonable backflow , the arteriotomy was closed . the patient 's leg continued to deteriorate post - operatively , with loss of sensation and further loss of power over 48 hours . ct peripheral angiography revealed an occlusion in the distal superficial femoral artery ( sfa ) . the right femoral vein was 4.2 mm greater in diameter compared to the same point in the left leg ( fig . the patient returned to theatre where an on - table angiogram showed an occluded sfa in the mid part . retrograde thrombectomy was completed through this incision to the sfa with subsequent angioplasty and stenting . though the sfa was patent , no blood flowed into the distal popliteal artery . a second on table angiogram was performed through the groin incision revealed an unusual finding a connecting fistula from the popliteal artery , filling the femoral vein , with lack of flow to the distal patent popliteal artery ( fig . further dissection to the posterior aspect of the knee revealed a popliteal arteriovenous fistula , with all the blood from the artery draining directly into the vein . endovascular stenting was not performed as the fistula was at the level of the joint . following closure of the fistula , with repair of the popliteal artery and vein , normal flow was achieved into the anterior tibial artery , which was the only available outflow vessel . had prolonged ischaemia , she was left with significant motor deficit in the limb . at six weeks follow - up , the fasciotomy sites were healing well with the aid of vacuum assisted closure . infection and malignancy can also predispose to fistula formation . a congenital arteriovenous fistula of the lower limb it can occur in syndromes such as klippel - trenaunay syndrome , or spontaneously , as is the apparent case with our patient . should the condition develop before closure of the epiphyses , there may be an increase in leg length . in retrospective measurement of the lower limbs six weeks post - operatively , her right leg was 3 cm longer than the left leg . there was also a discrepancy in the leg circumference , with the right leg measuring 7 cm greater than the left . our patient reported chronic venous hypertensive change in her right calf over her lifetime , with an enlarged right calf compared to the left side . other general physical findings that may be present include a palpable thrill and increased skin temperature . there may be a continuous machinery murmur on auscultation . bragman 's sign , whereby the heart rate drops after occluding the artery proximal to the fistula . the presence of an avf is demonstrated by low - resistance flow in the enlarged afferent artery , high velocity flow spectrum at the fistula site and a high velocity , arterialized waveform in dilated , thick - walled draining veins . although not 1st line , ct angiography may also be used to diagnose avfs . however , given the acute presentation of lower limb ischaemia , the patient did have a pre - operative angiogram , as urgent intervention was indicated . if indeed a pre - operative angiogram had been performed prior to initial operative management , there may have been a more favourable outcome , as the fistula may then have been detected on the primary intervention , which would have reduced the ischaemic injury to the limb . surgical repair has long been the mainstay of treatment , however there are other methods including ultrasound - guided compression that may be useful in the treatment of early fistulas . our patient was not an ideal candidate for endovascular repair as the fistula was located across a joint . ultrasound guided compression therapy was also inappropriate as the fistula in this case was longstanding . newer techniques of repair include percutaneous coil of the avf or embolization with n - butyl - cyanoacrylate . one case report described an incidental finding of a popliteal arteriovenous fistula , which appeared to be spontaneous , however that case the patient had bilateral venous hypertensive change . should a patient present with acute lower limb ischaemia and limb discrepancy or signs of asymmetrical venous hypertension , then routine intra - operative on - table angiogram should be performed after successful thrombectomy to exclude av fistula that may be managed with immediately endovascular intervention , to avoid further intervention . 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

vaccination against vaccine - preventable diseases is an essential component of women 's primary and preventive health care . to provide the best care for our patients , obstetrician - gynecologists are increasingly called to be vaccinators . a system is in place in this country to track adverse events following vaccine administration : the vaccine adverse event reporting system ( vaers ) . vaers is a voluntary reporting system coadministered by the centers for disease control and the food and drug administration . established in 1990 , vaers monitors vaccine safety and accepts adverse event ( ae ) reports following receipt of any us licensed vaccine . however , because reporting to vaers is not specifically mandated , utilization of vaers is necessarily dependent on familiarity with the existence of this system . we hypothesized that experience and familiarity with vaers are not common among obstetrician - gynecologists . to test this hypothesis and determine more specific knowledge deficits , we conducted a survey to assess the familiarity of practicing obstetrician - gynecologists with vaers . in this paper , we report the findings of this ten - question assessment . we invited a total of 1,000 practicing ob - gyns who are members of the american college of obstetricians and gynecologists ( acog ) to participate . three hundred recipients were members of the collaborative ambulatory research network ( carn ) , a group of physicians who have agreed to participate in 3 to 4 research surveys per year [ 2 , 3 ] . these 700 were chosen by systematically dividing the membership into subgroups of 100 that did not differ in distribution of age , gender , and geographic region and randomly selected 7 groups to participate in this study . after the last reminder mailing , a brief letter was sent to nonresponders that included three of the survey questions . the purpose of the letter was to assess whether there were differences between responders and nonresponders on key questions . the survey included the following demographic questions : gender , year of birth , year completed training , state of practice , location of practice , practice setting category , primary medical specialty , race / ethnicity , and primary race / ethnicity of patients . a total of ten questions were asked , nine knowledge questions and one question about the familiarity with vaers . question 1 asked about physicians ' familiarity with vaers ( in terms of prior use ) , and question 2 asked physicians to indicate what the purpose of vaers is . questions 3 , 4 , and 5 asked responders to indicate who sponsors vaers , the primary objectives of vaers , and who can report to vaers . questions 6 and 7 asked respondents to indicate if they are legally obligated to report an adverse vaccine event to vaers and what can be reported to vaers . question 8 asked physicians to indicate which of seven listed possibilities are recognized limitations of vaers . question 9 asked whether the adverse event rate for a vaccine can be calculated using vaers . the final question asked physicians to select which options were true regarding followup after a report is filed with vaers . a copy of the survey and the correct answers is included in the survey below . to examine whether those who had responded to the survey might be more knowledgeable or interested in vaers than those who did not , we sent a letter with three of the study questions to all of the survey recipients who had not returned a survey . the letter included year of birth , gender , and questions 1 , 7 , and 8 . vaers survey please give an overall assessment of your familiarity with the vaccine adverse event reporting system ( vaers ) ( please check one ) : have used vaers before ( 9.2% ) have not used vaers , but am familiar with its purpose ( 73.7% ) have not heard of vaers ( 17.1% ) what is the vaccine adverse event reporting system ( vaers ) ? ( please check one ) : post marketing safety surveillance program ( 90.5% ) online registry developed by anti - vaccine activists ( 0.9% ) pre - licensure adverse event reporting system ( 2.4% ) adverse event compensation program ( 3.7% ) who sponsors vaers ? ( please select all that apply ) : cdc ( 46.2% ) nih ( 13.5% ) fda ( 46.5% ) private industry ( 21.7% ) what are the primary objectives of vaers ? ( please select all that apply ) : detect unusual or rare vaccine adverse events ( 86.5% ) monitor increases in known adverse events related to a vaccine ( 67.9% ) identify patient risk factors for particular types of injuries ( 46.8% ) identify vaccine lots associated with increased reported adverse events ( 65.1% ) who can report to vaers ? ( please select all that apply ) : physicians ( 97% ) nurses ( 81% ) pharmacists ( 62% ) vaccine recipient ( 62% ) manufacturers ( 58% ) vaccine recipient spouse ( 28% ) true / false : if an adverse event occurs with a vaccine , the physician is legally required to report this adverse event to vaers ? ( please check one ) : 5.2% did not answer true ( 34.3% ) false ( 60.6% ) which is the best statement regarding what can be reported to vaers ? ( please check one ) : any adverse event ( 65% ) only those adverse events suspected to be vaccine - related ( 31.2% ) only those adverse events that require medical attention ( 2.4% ) only those adverse events that require hospitalization ( .3% ) what are recognized limitations to vaers data ? ( please select all that apply ) : dose not determine causality ( 66.0% ) underreporting of adverse events ( 84.4% ) increased reporting in the first few years after vaccine licensure ( 38.4% ) increased reporting after documentation of a known or alleged injury ( 41.9% ) reporting of coincidental events ( 63.2% ) data is not complete or necessarily accurate ( 64.1% ) can not calculate rates of adverse events ( 40.3% ) true / false : one can calculate the adverse event rate for a vaccine using vaers ? ( please check one ) : 6.7% did not answer true ( 21% ) false ( 72.8% ) after a report is filed with vaers , which of the following are true ? ( please select all that apply ) : no follow up is available ( 5.8% ) vaers staff may request additional information ( 85.3% ) patient consent is not required for release of medical records ( 20.5% ) medical records sent to vaers become public record documents ( 6.1% ) . note that correct answers are in italic with ( % ) of responders who chose each option . please give an overall assessment of your familiarity with the vaccine adverse event reporting system ( vaers ) ( please check one ) : have used vaers before ( 9.2% ) have not used vaers , but am familiar with its purpose ( 73.7% ) have not heard of vaers ( 17.1% ) have used vaers before ( 9.2% ) have not used vaers , but am familiar with its purpose ( 73.7% ) have not heard of vaers ( 17.1% ) what is the vaccine adverse event reporting system ( vaers ) ? ( please check one ) : post marketing safety surveillance program ( 90.5% ) online registry developed by anti - vaccine activists ( 0.9% ) pre - licensure adverse event reporting system ( 2.4% ) adverse event compensation program ( 3.7% ) post marketing safety surveillance program ( 90.5% ) online registry developed by anti - vaccine activists ( 0.9% ) pre - licensure adverse event reporting system ( 2.4% ) adverse event compensation program ( 3.7% ) who sponsors vaers ? ( please select all that apply ) : cdc ( 46.2% ) nih ( 13.5% ) fda ( 46.5% ) private industry ( 21.7% ) private industry ( 21.7% ) what are the primary objectives of vaers ? ( please select all that apply ) : detect unusual or rare vaccine adverse events ( 86.5% ) monitor increases in known adverse events related to a vaccine ( 67.9% ) identify patient risk factors for particular types of injuries ( 46.8% ) identify vaccine lots associated with increased reported adverse events ( 65.1% ) detect unusual or rare vaccine adverse events ( 86.5% ) monitor increases in known adverse events related to a vaccine ( 67.9% ) identify patient risk factors for particular types of injuries ( 46.8% ) identify vaccine lots associated with increased reported adverse events ( 65.1% ) who can report to vaers ? ( please select all that apply ) : physicians ( 97% ) nurses ( 81% ) pharmacists ( 62% ) vaccine recipient ( 62% ) manufacturers ( 58% ) vaccine recipient spouse ( 28% ) vaccine recipient ( 62% ) manufacturers ( 58% ) vaccine recipient spouse ( 28% ) true / false : if an adverse event occurs with a vaccine , the physician is legally required to report this adverse event to vaers ? ( please check one ) : 5.2% did not answer true ( 34.3% ) false ( 60.6% ) which is the best statement regarding what can be reported to vaers ? ( please check one ) : any adverse event ( 65% ) only those adverse events suspected to be vaccine - related ( 31.2% ) only those adverse events that require medical attention ( 2.4% ) only those adverse events that require hospitalization ( .3% ) any adverse event ( 65% ) only those adverse events suspected to be vaccine - related ( 31.2% ) only those adverse events that require medical attention ( 2.4% ) only those adverse events that require hospitalization ( .3% ) what are recognized limitations to vaers data ? ( please select all that apply ) : dose not determine causality ( 66.0% ) underreporting of adverse events ( 84.4% ) increased reporting in the first few years after vaccine licensure ( 38.4% ) increased reporting after documentation of a known or alleged injury ( 41.9% ) reporting of coincidental events ( 63.2% ) data is not complete or necessarily accurate ( 64.1% ) can not calculate rates of adverse events ( 40.3% ) dose not determine causality ( 66.0% ) underreporting of adverse events ( 84.4% ) increased reporting in the first few years after vaccine licensure ( 38.4% ) increased reporting after documentation of a known or alleged injury ( 41.9% ) reporting of coincidental events ( 63.2% ) data is not complete or necessarily accurate ( 64.1% ) can not calculate rates of adverse events ( 40.3% ) true / false : one can calculate the adverse event rate for a vaccine using vaers ? ( please check one ) : 6.7% did not answer true ( 21% ) false ( 72.8% ) after a report is filed with vaers , which of the following are true ? ( please select all that apply ) : no follow up is available ( 5.8% ) vaers staff may request additional information ( 85.3% ) patient consent is not required for release of medical records ( 20.5% ) medical records sent to vaers become public record documents ( 6.1% ) . no follow up is available ( 5.8% ) vaers staff may request additional information ( 85.3% ) patient consent is not required for release of medical records ( 20.5% ) medical records sent to vaers become public record documents ( 6.1% ) . , participants were grouped into two groups ( answered correctly or answered incorrectly ) , and we computed the percent of respondents answering correctly . we assessed differences in age and gender between those who answered the question correctly and those who did not . for questions without a correct answer ( which include only one question about familiarity with vaers ) , we analyzed differences in age and gender among the response options . in obstetrician - gynecologists , age and gender are highly associated . hence , when gender differences were assessed , we controlled age using a dichotomous variable . participants were grouped using a median split into two roughly equal sized groups : physicians born between 1933 and 1958 ( 51% , n = 188/372 ) and physicians born between 1959 and 1979 ( 49% , n = 184/372 ) . separate tests for differences between males and females were run for the older age group and the younger age group . data were analyzed using a personal computer based version of spss 17.0 ( spss inc . , one - way analysis of variance was used for continuous variables ; analyses were conducted for categorical variables . the response rate for carn participants was 57% ( 171/300 ) . for non - carn participants , the response rate was 32.1% ( 225/700 ) . overall , a total of 397 physicians responded to the survey ( 39.7% response rate ) . twenty responders were considered ineligible and were eliminated from data analysis ( i.e. , 9 physicians indicated being retired , and 11 physicians returned the survey blank ) , leaving 377 eligible responders ( 167 carn , 209 non - carn , and one unidentified ) . of the 377 eligible responders , 50 did not complete the survey and were excluded from analysis , resulting in a total sample of 327 obstetrician - gynecologists . we found no significant differences between carn and non - carn responders ( data not shown ) ; therefore , we collapsed the data and analyzed in aggregate form . average year of birth was 1958 ( 9.7 ) , and male physicians ( 1954 9.4 ) were significantly older than female physicians ( 1962 8.3 ) ( f(1 , 318 ) = 67.3 , p < .001 ) . white , non - hispanic was the most common race of physicians ( 82% ) and their patients ( 72% ) . around half ( 54% ) were in group practice and 73% practice general obstetrics and gynecology . overall , only one respondent answered all nine knowledge questions correctly . when asked about the sponsors of vaers , only 12.5% correctly indicated that the cdc and fda are the sponsors and that nih and private industry are not . a total of 24.2% of the sample correctly indicated all six of the individuals listed can report to vaers . when asked what happens after a report is filed , 17.4% correctly indicated that vaers staff could request additional information and that patient consent is not required . the number of recognized limitations to vaers identified by each physician was summed for a total limitations score . differences in age were found among females only ; older female physicians had a higher mean limitations score ( m = 4.6 , sd = 2.0 ) than younger female physicians ( m = 3.9 , sd = 2.1 ) ( f(1 , 161 ) = 4.4 , p = .038 ) . gender differences were found on some of the survey questions . as shown in figure 1 , males were twice as likely to be not familiar with vaers than females , with 23% of males indicating not familiar compared with 11% of females ( = 10.7 , p = .005 ) . when assessing for differences in gender among younger and older physicians , males were more likely to indicate not familiar than females in the older age group ( = 8.6 , p = .014 ) , but not in the younger age group . as shown in figure 2 , females were more likely than males to indicate correctly all four objectives of vaers ( 40% of males versus 52% of females , = 4.2 , p = .041 ) . when assessing for differences in gender among younger and older physicians , females were more likely to indicate the correct vaers objectives in the older group ( 39% of males versus 61% of females , = 6.5 , p = .01 ) , but there was no significant difference between males and females in the younger group . of the 77 , 12 were retired or did not complete over half of the questions on the letter and were therefore excluded . we compared the survey responses with letter responses to assess potential differences between those who responded to the survey and those who did not respond . the number of eligible letter responders was 65 , with 18 from the carn group and 47 from the non - carn group . letter responders were not significantly different from survey responders in gender or year of birth . forty - three percent were female , 46.2% were male , and 10.8% did not identify their gender . we found no significant differences between survey responders and letter responders on any of these questions . this suggests that the answers we received to the survey are similar to those we would have received even if we had had a larger response rate . safety concerns are one of the most common immunization concerns cited by patients . in fact , because of potential impact on the fetus , in our specialty , safety concerns can be amplified both for patients and providers for immunizations recommend during pregnancy [ 57 ] . we have seen that data generated from vaers is able to reinforce the safety profile for recommending vaccines during pregnancy , including h1n1 and tdap [ 810 ] . with increased reporting to vaers , the data base will become more robust . currently , most obstetrician - gynecologists acknowledge familiarity with vaers . however , only one respondent correctly answered all the questions correctly and few ( 9% ) obstetrician - gynecologists surveyed have used vaers . hence , we verified our original hypothesis that there is a lack of familiarity with vaers in the ob / gyn 's professional community . to determine more specific information about knowledge deficits , we stratified the answers to this survey based on age and gender . when we compared age differences , older physicians were less familiar with vaers in general and with the specific objectives of vaers in particular . for controlling age , we stratified the data by gender and found that women were more likely than men to state that they were aware of vaers and also to know all the objectives of vaers . when stratifying both by age and gender , older women were more likely to know the correct answers to vaers objectives than older males , but in the younger group , the gender differences were not seen . this study does not address attitudinal or practice differences in immunization administration nor were we able to find other studies documenting the frequency of immunization administration in obstetrician - gynecologist practices based on provider age or gender . however , studying these differences could provide useful insight into how we see ourselves or practice as providers of immunizations and allow more targeted cme strategies . this survey addresses knowledge and use of vaers . as a survey with a response rate of 37% and a sample size of 1000 those who responded to the survey may be more likely to have knowledge or interest in vaers . we tested this by sending all nonresponders a brief letter soliciting responses to three of the survey questions . when comparing survey responders and letter responders , we did not see differences in familiarity with vaers and prior use of vaers supporting the generalizability of our survey . while some differences may be present between responders and nonresponders , these differences may not be as important as the similar low prior use of vaers between these two groups . regarding obstetrician - gynecologist use and knowledge of vaers , the message is simple and clear : it could be better . we can be encouraged that about 50% of eligible pregnant patients received the flu vaccine in 2011 ( compared to 17% prior to the h1n1 pandemic ) . however , we can utilize a multifactorial approach to further improve the knowledge about and integration of immunizations into the ob - gyn 's practice . the american congress of obstetricians and gynecologists ( acog ) has launched a website , which provides scientific and practical information to facilitate integration of immunizations into clinicians ' offices . also , cme courses are commonly offered at acog 's annual clinical meeting for immunizations . residents ' education on immunizations is now a requirement . in areas of the country where a vaccine preventable epidemic has occurred , such as the recent epidemic of pertussis in the state of washington , pregnant patients have been directly targeted and encouraged to solicit immunizations from their providers . adding immunization questions to written and oral board specialty certification examinations as well as the written recertification examination would also necessitate an immunization fund of knowledge for practicing ob - gyns . specific to vaers , haber and colleagues have recently demonstrated the value of internet - based reporting as a means to improve provider utilization of this safety monitoring system . this survey demonstrates that familiarity , understanding , and use of the vaers data base merit improvement . further research comparing attitudes and practice patterns of obstetrician - gynecologists regarding immunization is also merited so that we can strategically implement education efforts to enhance obstetrician - gynecologist 's utilization of vaccines : a proven primary prevention tool .

compared to hiv - uninfected individuals living with mycobacterium tuberculosis infection ( mtbi ) , people living with hiv ( plhiv ) possess a 2037-fold higher risk of developing tuberculosis ( tb ) . this disease accounts for approximately 25% of all hiv - related deaths [ 13 ] . the prevailing paradigm explaining the immunologic mechanism underlying this epidemiologic phenomenon is that hiv eliminates cd4 t cells , including those presumed to be essential for mtb control . interferon - gamma ( ifn- ) production by t cells in response to an mtb stimulus is the standard immunologic test for mtbi ; however , this measurement does not differentiate mtbi from tb disease . since progression along the tb disease spectrum is a major predictor of mortality for plhiv , there is a critical need for clinical immune biomarkers that reliably predict a patient 's precise tb condition . multiparametric analyses of the immune system have illuminated our insight of the mechanisms of disease pathogenesis and protection [ 410 ] . it is clear now that the immune response to any given pathogen is complex and multifaceted . thus , relying on a single immunologic measure to yield an accurate diagnosis is suboptimal and perhaps even misleading . understanding the dynamics of an immune equilibrium between competing responses may improve our predictive capacity for clinical outcome ( see supplemental figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/1478340 ) . for example , during tb infection , anti - inflammatory agents ( such as il-4 , il-10 , and immune checkpoint inhibitors ) prevent host tissue destruction due to excessive cell - mediated immunity [ 1114 ] . in contrast , excessive il-4 or il-10 production may result in a decreased containment of intracellular mtb and therefore theoretically increased risk of tb disease progression [ 15 , 16 ] . to evaluate if the balance between pro- and anti - inflammatory cd4 t cell responses from plhiv subjects with tb correlates with tb progression , we quantified the production of mycobacteria - specific ifn- , il-4 , and il-10 simultaneously . we hypothesized a direct relationship between hiv disease severity and a skewing of the immune equilibrium away from cell - mediated and towards an anti - inflammatory profile . in addition , we profiled the cytokine responses from cd8 , cd56cd3 nk cells , and cd3cd4cd8cd56 ( double negative t lymphocytes ) . as the cd4 t cell compartment wanes with advancing hiv disease status , these immune cell types gain prominence . for plhiv at risk for tb disease development , our objective is to elucidate the effect of hiv progression on the balance of the antimycobacterial immune response . adults with microbiologically confirmed ( genexpert mtb / rif or culture positive ) pulmonary tb ( n = 12 ) were enrolled after completing written informed consent . ethical approval was obtained by all relevant bodies including baylor college of medicine , swaziland children 's foundation , baylor college of medicine irb , and the swaziland ethics committee . blood collected into acd tubes was transported to the laboratory , within 4 hours , and pbmcs were isolated using ficoll density gradient as previously described . to elicit an immune response by the adaptive and innate immune responses , pbmcs were stimulated with mycobacterium bovis whole cell lysate ( 5 g / ml ) for 16 hours which was obtained through bei resources , naid , nih ( mycobacterium bovis , strain af 2122/97 atcc baa-935 , whole cell lysate , nr-31211 ) . stimulation occurred in the presence of brefeldin a ( sigma , 1 g / ml ) , golgi - stop ( bd biosciences , 0.7 g / ml ) , and costimulatory antibodies cd28/cd49 ( bd biosciences , 2.5 g / ml ) . following stimulation , cells were washed and stained for viability ( ghost dye , tonbo biosciences ) , surface antigens , cd3 ( biolegend ) , cd8 ( biolegend ) , cd4 ( ebioscience ) , cd14 ( biolegend ) , and cd56 ( biolegend ) , and intracellular proteins , ifn- ( life technologies ) , il-4 ( bd bioscience ) , and il-10 ( bd bioscience ) . the cells were acquired on a bd lsr ii fortessa flow cytometer as previously described . flow cytometry and statistical analyses were performed using flowjo x ( treestar ) and graphpad 6.0 ( graphpad software ) , respectively . while cd4 t cell ifn- production remains the dogmatic measurement of mycobacterial immunity , it fails to predict which individuals are at increased risk of disease progression . to this end , we hypothesized a more accurate means via a comprehensive immune profile balancing the anti - inflammatory cytokines ( il-4 and il-10 ) compared to inflammatory ones ( ifn- ) . we measured the mycobacteria - specific il-4 : ifn- and il-10 : ifn- ratios from cd4 , cd8 , cd56 , and double negative ( cd3cd4cd8cd56 ) cells . representative dot plots are available in figures 1(a ) and 1(b ) and the gating strategy is available in supplemental figure 2 . as a proxy for hiv progression , we calculated the cd4 : cd8 ratio . participants had a mean age of 24 ( range 1638 ) , 58% female with 100% on antiretroviral therapy ( art ) ( see table 1 ) . as hiv progresses and the percentage of cd4 cells decreases , the percentage of lymphocytes that are cd8 or dn increases ( figure 1(f ) ) ; this association was not seen with cd56cd3 nk cells . similar to previous descriptions , the percentage of cd4 t cells producing mycobacteria - specific ifn- did not correlate with cd4 : cd8 ratio ( spearman r = 0.059 ; p = 0.83 ) ( figure 1(c ) ) , while the percentage of il-4 producing cd4 cells increased with hiv progression ( figure 1(d ) ) . when considering the balance , il-4 : ifn- increased as the cd4 : cd8 ratio decreased ( spearman r of 0.596 ; p = 0.02 ; figure 1(e ) ) . the correlation of increasing il-4 : ifn- ratio with decreasing cd4 : cd8 ratio occurred in both the adaptive ( cd4 and cd8 ) and innate ( cd56cd3 nk cell ) response as well as in the immune response of double negative ( dn ) lymphocytes ( figure 1(e ) ) . while there are many cells capable of producing il-10 ( lymphocytes , granulocytes , monocytes , macrophages , and dendritic cells ) , their production occurred predominantly in nonlymphocytes ( figure 2(a ) ) . in contrast , the contribution from cd56 nk or cd3 t cells was minimal ( figure 2(a ) ) . therefore , we correlated the il-10 production from nonlymphocytes to the total percentage of lymphocytes producing mycobacteria - specific ifn- as an appropriate measure for the balance of pro- and anti - inflammatory immune responses ( figure 2(a ) ) . as hiv disease progressed ( as measured by declining cd4 : cd8 ratio ) , the nonlymphocyte il-10 : lymphocyte ifn- ratio increased ( spearman r = 0.76 ; p = 0.01 ) ( figure 2(b ) ) . therefore , while the percentage of cd4 t cells producing ifn- did not correlate with hiv progression , the balance of anti - inflammatory ( il-4 or il-10 ) and cell - mediated immune responses did . combatting the hiv - tb epidemics ravaging vulnerable populations is going to require significant investments in clinical care and also cutting edge basic and translational research to discover new diagnostics , identify pathways for immunotherapeutics , and develop effective vaccine platforms . here , amongst a cohort of swaziland plhiv with tb disease , we show that the benefit of applying a comprehensive and balanced profile of antimycobacterial immunity is of paramount importance . for progression to tb disease , hiv infection is the greatest risk factor [ 13 ] and , therefore , identification of immune correlates of tb progression amongst plhiv is of utmost importance . the central doctrine of mycobacterial immunity relies on cd4 t cell ifn- production ; however , this measurement fails to differentiate infection from disease , predict those at high risk for progression , or predict vaccine immunogenicity [ 17 , 18 ] . thus , there is an urgent need for reliable immune biomarkers of protection , as well as disease advancement with recent research delineating the roles of cytotoxic t lymphocytes ( cd8 ) [ 4 , 20 ] , nk cells , double negative cells , monocytes , and macrophages . in contrast to the classic approach of only evaluating the cell - mediated mycobacterial immune response , we demonstrate how the simultaneous evaluation of cell - mediated and anti - inflammatory responses improves our understanding of the immune processes at play . in contrast to the premise that ifn- defines mycobacterial immunity best , we show that as hiv disease progresses , both adaptive and innate lymphocyte lineages polarize towards an il-4-dominated anti - inflammatory response . the evidence for hiv promoting a th2 skewing of the immune response is controversial as it is dependent on the methodology ( pcr , elisa [ 22 , 23 ] , or flow cytometry ) and type of stimuli ( antigen - specific versus pmai / i ) . here , using multiparametric flow cytometry to measure the antigen - specific response , we show that hiv progression causes an increase in the cd4 t cell il-4 : ifn- ratio . for the first time to our knowledge , we demonstrate a negative correlation between il-4 and ifn- that occurs amongst not only cd4 t cells , but also cd8 t cells , double negative lymphocytes , and nk cells . these preliminary findings raise interesting questions about the pathophysiology of tb disease progression in the hiv setting . one possible explanation for the increased il-4 : ifn- ratio may be increased cell turnover . gata-3 , the master transcription factor promoting il-4 and inhibiting ifn- , is required for thymic development . it is plausible that as hiv progression results in high lymphocyte turnover , the increasing percentage of gata-3 nave immune cells explains the etiology of il-4 skewing . if this were the explanation , one would expect the ratio to normalize the longer an individual is on suppressive art . our data suggest that cd3 t cells do not produce significant pathogen - specific il-10 . in contrast , the ratio of nonlymphocyte il-10 : lymphocyte ifn- correlates inversely with hiv progression . animal models [ 12 , 14 , 26 , 27 ] suggest that an appropriate balance of anti - inflammatory to proinflammatory immunity is needed to control intracellular mtb . correlation of these animal models with longitudinal human data is needed to identify the ratio at which il-10 is protective versus when it becomes detrimental to the protective cell - mediated response . these preliminary findings in this communication are significant insofar as they begin to define the relationship between hiv disease severity and the immune response equilibrium amongst multiple lymphocyte lineages . replication of these primary findings in longitudinal cohorts of tb exposed plhiv promises to assess the predictive utility il-4 : ifn- or il-10 : ifn- ratios , further elucidating the mechanisms of mtb protective immunity and therefore supporting development of targeted immune therapies .

congenital or primary hypoparathyroidism presenting with new onset seizures in an adult is an infrequent phenomenon . there is a tendency for solo practitioners in india to feel apprehensive about adult patients presenting with new onset seizures . the immediate response is to give an injectable anti - epileptic drug and refer without considering further evaluation . it is important to have an approach to adult , first onset seizure in family practice . it would benefit the patient and it would in the long term earn respect for the family practitioner in the community . the following case report highlights how looking for causes of first onset seizures enabled the diagnosis of congenital hypoparathyroidism to be made , a condition which is treatable and reversible , in a primary care family practice . a 29-year - old female patient presented to our urban health center with a history of recent onset abnormal movements of upper limb and eyes for the duration of 2 months . she did not have previous history of seizures , but her family members noted the abnormal movements occurred on standing . these abnormal movements were not associated with tongue biting , up rolling of eyes or loss of consciousness . she had been to local practitioners who had started her on anti - epileptic drugs and anxiolytics . she also had a history of treatment for tuberculous lymphadenitis at the age of 13 years . a documented treatment for carpopedal spasm was noted during the treatment for tuberculous lymphadenitis in the local dots center . she had not been investigated earlier for the early onset of cataract or for the carpopedal spasms . she is the second of four siblings and was unmarried due to episodes of weakness as perceived by her family . there was no family history of seizures , diabetes , hypertension , thyroid disorders or any other endocrine disorders . physical examination revealed a thinly built woman , weight of 45 kg , blood pressure of 110/60 mmhg in right upper limb at sitting position , pulse rate of 112/min . she did not have pallor , icterus , cyanosis , pedal edema or generalized lymphadenopathy . she had a mature cataract in her left eye and intra - ocular lens was in situ in the right eye . she was supported to walk into the consultation room and on standing developed abnormal posturing ( pedal spasms ) during which time she was conscious and conversing with the examining physician . the investigations showed : hemoglobin : 12.4 g%serum calcium : 5.4 mg% ( normal values : 8.3 - 10.4 mg%)serum phosphorous : 5.6 mg% ( normal values : 2.5 - 4.6 mg%)thyroid stimulating hormone : 1.54 miu / mlserum cortisol , random : 13.56 g%serum parathyroid hormone : <3 pg / ml ( normal values : 8.0 - 74 pg / ml ) . serum calcium : 5.4 mg% ( normal values : 8.3 - 10.4 mg% ) serum phosphorous : 5.6 mg% ( normal values : 2.5 - 4.6 mg% ) thyroid stimulating hormone : 1.54 miu / ml serum cortisol , random : 13.56 g% serum parathyroid hormone : <3 pg / ml ( normal values : 8.0 - 74 pg / ml ) . her blood sugar levels , renal function tests , other serum electrolytes and liver function tests were normal . an electroencephalography ( eeg ) was not done since the history and presence of seizures were convincing . on initial clinical evaluation , the diagnosis of metabolic disorder was considered because of the presentation of cataract in young age and carpopedal spasms . the diagnosis of primary hypoparathyroidism was made after review of the initial investigations and in consultation with the endocrinologist in the tertiary hospital to which the family practice center is attached . as the diagnosis was clear and there were no benefits to be gained , further imaging was deferred . she was started on monthly injections of vitamin d3 ( cholecalciferol ) and daily oral calcium and vitamin d3 tablets amounting to 2.4 g of calcium and 500 iu of vitamin d3 per day . her serum calcium ( 8.5 mg% ) and phosphate ( 3.1 mg% ) became normal in 2 months and continues to be normal . the plan for continuity of care was to administer monthly injections of vitamin d3 lifelong with oral calcium tablets and monitor urine calcium once a year to monitor for risk of renal stones . the incidence of new adult onset seizures is not well documented in india . however , a community based study states that the average annual incidence rate of epilepsy is 42.08 per 100,000 per year . a longitudinal study done in an urban population however quotes the average annual incidence rate to be 27.3 per 100,000 per year . seizures account for 2.1% of adult emergency department visits in a tertiary hospital in india . the number of patients who present with seizures to a general practitioner ( gp ) in india is not documented even though gp is usually the first health worker who sees the patient with seizures . there are currently practice guidelines for first onset seizures in adults available in developed countries but not one uniformly accepted and practiced in india . as a result many adults who develop first onset seizures are managed symptomatically , but reversible causes are not investigated or looked for . there is also a lack of continuity of care for these patients . in primary care clinical practice , the work up of any adult who presents with history of seizures consists of two processes : classifying the seizure type and determining the causes . they have to initially be classified whether the seizures were , generalized or focal or absent . eeg is helpful only even the history of seizure itself is not convincing or in absence seizures . when an adult patient presents with first onset of seizures an accurate history from patient and/or family members and the eye witnesses should be elicited . a meticulous physical examination will often point to a cause , as in this patient who had mature cataract in an eye and a history of cataract removal in the other eye . partial or focal seizures need imaging studies and an infective workup as early as possible . according to the guidelines for the management of epilepsy in india imaging is indicated in focal seizures and in evaluation of seizures in acute situations like head injury , cerebrovascular injury or encephalitis . for those with generalized seizures , investigations for possible provoked seizures are serum electrolytes including calcium , liver function tests , renal function tests , infective screen ( lumbar puncture ) if indicated and breath test for alcohol . as this patient had severe hypocalcemia she was investigated for endocrine abnormalities . in the management of this patient , since a step - wise approach was used , many fancy and routine investigations such as scans and eegs were avoided . a step wise approach to a person with adult onset seizures , which incorporates meticulous history , a detailed physical examinations and relevant investigations , will help the family physicians to manage these patients .

the selection of an optimal treatment modality for patients with n2-positive stage iiia ( n2-iiia ) non - small cell lung cancer ( nsclc ) has been a major controversial issue in thoracic oncology . a multimodality approach rather than a single modality therapy has been preferred , and the current national comprehensive cancer network ( nccn ) guideline recommends definitive concurrent chemoradiotherapy ( ccrt ) as the category 1 option , while induction chemotherapy , with or without radiation therapy ( rt ) , and surgical resection is the alternative option . the recent practice guidelines from the american college of chest physicians ( accp ) primarily recommends either definitive ccrt or induction therapy followed by surgery in the patients with discrete n2-iiia nsclc over either surgery or rt alone . both guidelines have multimodality treatment in common , and the accp guideline recommends that the treatment plan be made with input from a multidisciplinary team . a multidisciplinary lung cancer team was established at samsung medical center ( smc ) when it was opened in 1994 . since then , smc s lung cancer team has actively implemented decision - making during lung cancer therapy , which has consisted of a full spectrum of lung cancer diagnoses and treatments including pulmonology , thoracic surgery , radiation oncology , medical oncology , diagnostic radiology , pathology , and nuclear medicine . because of frequent locoregional failures following definitive ccrt without surgery , we have incorporated surgical resection following preoperative ccrt since 1997 , and this trimodality therapy strategy has been the primary policy at smc during treatment of patients with n2-iiia nsclc . following trimodality therapy , the authors reported that the 5-year progression - free survival ( pfs ) and overall survival ( os ) rates were 26.9% and 43.3% , respectively . although this strategy has demonstrated improved and favorable locoregional control and survival outcomes , surgical resection can not be undertaken following preoperative ccrt in some patients . first , treatment policy changes from trimodality to bimodality therapy during or after the preoperative ccrt course , and second , bimodality therapy is primarily recommended from the onset of treatment . typical patient factors necessitating a change in treatment policy include poor to marginal cardio - pulmonary function status , medical comorbidity , and the patient s refusal of surgical resection . typical disease factors leading to such a change include extensive and infiltrative nature of the tumor and/or lymph nodes that are judged to impede curative resection or necessitate pneumonectomy rather than lobectomy . the purpose of this study was to evaluate the treatment outcomes following bimodality therapy in patients with n2-iiia nsclc at smc . from may 1997 to december 2012 , a total of 633 patients were diagnosed as having n2-iiia nsclc at smc based on the american joint committee on cancer ( ajcc ) seventh edition cancer staging manual ( fig . 528 patients ( 83.4% ) were judged to be medically operable with resectable or marginally resectable disease , while 105 ( 16.6% ) were either medically inoperable or had unresectable disease because of the extensive and infiltrative nature of the tumor and/or lymph nodes . according to the smc lung cancer center treatment policy , however , the planned preoperative ccrt course could not be completed in 51 patients ( 9.7% ) due to disease progression in 22 ( 4.2% ) , patient refusal in 17 ( 3.2% ) , treatment - related mortality in six ( 1.1% ) morbidity in four ( 0.8% ) , and other co - morbidity in two ( 0.4% ) . after re - assessment of resectability following preoperative ccrt , 456 patients ( 86.4% ) underwent curative resection as initially planned , while the remaining 21 ( 4.0% ) , who had a presumed high risk of surgical resection or refused surgical resection , received boost ccrt ( bimodality group i , hereafter ) . among 105 patients who had unresectable disease at diagnosis , 61 who were not fit for aggressive treatment received either rt alone ( n=50 ) or chemotherapy alone ( n=11 ) . definitive ccrt was recommended to 44 patients as a primary therapy ( bimodality group ii , hereafter ) . after approval by the institutional review board ( 2013 - 08 - 085 ) , the medical and rt records of the 65 patients in bimodality groups i and ii were retrospectively reviewed . the median rt dose was 66 gy in 33 fractions using 4 - 10 mv photon beams generated by a linear accelerator ( varian medical systems inc . , palo alto , ca ) . a median treatment break of 36 days was imposed during the rt course for 13 patients in bimodality group i , mainly because judgment on resectability was made 4 to 6 weeks after preoperative ccrt . to compensate for this unintended treatment break , the total rt dose was intentionally escalated to a median of 72 gy in these patients . therefore , the mean rt dose was higher in bimodality group i ( p=0.002 ) . the most common chemotherapeutic regimen during rt was weekly paclitaxel or docetaxel plus cisplatin or carboplatin in 54 patients ( 83.1% ) , followed by cisplatin plus oral etoposide at 4-week intervals in 11 patients ( 16.9% ) . the primary endpoints of the current study were pfs and os , and the secondary endpoints were in - field locoregional control ( lrc ) and patterns of disease progression . the durations of observation were calculated from the first date of ccrt until the date of event , death or censoring . disease progression included any type of treatment failure , while in - field locoregional progression was defined as persistent or newly developing lesion within the actual rt target volume . neither out - field remote intrathoracic failure nor hematogenous lung metastasis were considered as locoregional progression . t tests were used to compare continuous variables such as age , forced expiratory volume in 1 second , tumor size , and radiation dose . chi - square or fisher exact tests were used to compare categorical variables between treatment groups . the rates of pfs , os , and in - field lrc were calculated using the kaplan - meier method and compared using the log - rank test . a p - value of 0.05 was considered statistically significant , and sas software ( ver . the median age of the 65 patients in both groups was 65 years ( range , 36 to 76 years ) , and the vast majority of the patients were male ( 59 , 90.8% ) and ex - smokers ( 60 , 92.3% ) . squamous cell carcinoma was the most common histology in 39 patients ( 60.0% ) , followed by adenocarcinoma in 22 ( 33.8% ) . based on initial imaging studies , which included chest - computed tomography scan and/or positron emission tomography scan , the median tumor size was 4.3 cm ( range , 1.0 to 11.6 cm ) , and ct1 - 2 stages were more common than ct3 ( 41 vs. 24 ) . the majority of the patients ( 60 , 92.3% ) had clinically evident n2-positive disease , and about half ( 33 , 50.8% ) underwent histologic confirmation . five patients ( 7.7% ) with clinically n2-negative disease were confirmed to have n2 disease based on histological analysis . the procedures for histologic confirmation of n2 disease were endobronchial ultrasound - guided transbronchial needle aspiration in 17 patients , mediastinoscopic biopsy in 11 , videoassisted thoracoscopic biopsy in two , endoscopic transesophageal ultrasound guided biopsy in one , bronchoscopic biopsy in one , and open excision of the lymph node in one . based on all clinical evaluation tools , 43 and 22 patients ( 66.2% and 33.8% , respectively ) were known to have single and multi - station n2 involvement , respectively . during the median follow - up duration of 18.8 months ( range , 1.6 to 173.1 months ) , the most common first treatment failure was distant metastasis , which was observed in 23 patients ( 34.8% ) , followed by locoregional progression in 16 ( 24.6% ) . additionally , 12 out of 16 patients with locoregional progression had failure within the rt volume . the most frequent distant metastatic organs were the brain in nine patients , followed by the adrenal gland in four . the median pfs was 18.8 months , and the 2- and 5-year pfs rates were 45.9% and 30.5% , respectively ( fig . a total of 39 patients ( 60.0% ) died during the follow - up period . pulmonary causes such as pneumonia , pneumonitis , and acute respiratory distress syndrome were responsible for seven deaths , which occurred at a median of 4.0 months ( range , 2.3 to 8.5 months ) . there were three intercurrent deaths from hepatocellular carcinoma , myocardial infarction , and gastrointestinal bleeding . the median os was 28.6 months , and the 2- and 5-year os rates were 50.1% and 33.3% , respectively ( fig . univariate analysis revealed that the cisplatin plus oral etoposide regimen was associated with significantly better pfs and os ( table 3 ) , and females tended to have a better os . there were no significant differences in survival outcomes between bimodality groups i and ii . other variables such as ct stage , involvement of multistation lymph nodes , and treatment break did not affect survival outcomes . n2-iiia nsclc involves potential heterogeneity in mediastinal lymph node involvement , which could influence the decision regarding the treatment plan among various options . a survey study showed that choice of treatment plan differed according to the extent of mediastinal involvement . in the case of single - node disease , 92% of the oncologists incorporated surgical resection into a treatment plan , while definitive chemoradiation was mostly preferred in the case of bulky multi - station n2 disease . in addition to the heterogeneity of n2 disease , there might also be heterogeneity in the physician s treatment plan preference . among patients who received trimodality therapy for n2-iiia nsclc at our institution , 66.8% and 36.6% had clinically evident and multi - station n2 disease , respectively . our institution has adopted a multidisciplinary approach to management of patients with lung cancer , and tri- or bi - modality treatment showed consistently favorable outcomes . although not all patients had histological confirmation of involvement of the n2 lymph node in this study , most were found to have clinically evident n2 disease upon imaging analyses , and about one - third of the patients had multi - station n2 disease . resectability was assessed before and/or during the course of ccrt , in accordance with the multidisciplinary team approach described above . our rationale for increasing radiation dose to compensate for the unintended treatment break applied in 13 patients in bimodality group i is very difficult to validate based on the current study results . twenty - one patients in bimodality group i , 13 of whom had treatment break and increased radiation dose , showed similar clinical outcomes to those in bimodality group ii ( table 3 ) . naturally , it is desirable to avoid or shorten the treatment break whenever possible ; however , when such a break can not be avoided , an effort to intensify the boost ccrt seems a reasonable approach . trimodality therapy that includes surgical resection following preoperative ccrt in physiologically fit patients with n2-iiia nsclc has long been the primary recommendation at smc , and subsequent clinical outcomes have recently been reported . definitive bimodality therapy has been recommended to physiologically fit patients who are poor candidates for surgical resection due to either increased surgical risk or patient refusal of trimodality therapy . direct comparisons of the outcomes following tri- and bimodality therapy might be biased owing to selection bias and heterogeneity in the patient characteristics in this retrospective study . a randomized controlled phase iii trial compared the two treatment modalities and determined that the median os was 23.6 months in patients who underwent surgical resection and 22.2 months in patients who underwent continued radiotherapy ( p=0.24 ) , which was similar to the results of the present study . the corresponding pfs were 12.8 and 10.5 months , respectively , in the two treatment groups ( p=0.017 ) . although no survival difference was observed between the two treatment modalities , a survival benefit was observed in patients who received lobectomy after neoadjuvant ccrt ( median os , 33.6 months vs. 21.7 months ; p=0.002 ) . decreased survival in pneumonectomized patients was suggested as the main reason for the limited survival benefit following trimodality treatment . improved survival after ccrt plus lobectomy and decreased survival after ccrt plus pneumonectomy negative survival influence following pneumonectomy has clinical implications in terms of treatment selection in patients with n2-iiia nsclc . pneumonectomy candidates appear to be more suitable for definitive bimodality therapy than trimodality therapy , while candidates who do not require pneumonectomy might benefit from trimodality therapy . treatment results after ccrt with cisplatin plus oral etoposide followed by consolidation chemotherapy were previously reported , and the median pfs and 2-year pfs rate were 12.3 months and 40% , respectively . because more than half of the patients in that study had n3 disease , only a small portion of patients with favorable prognosis should have been included . in addition , the role of the docetaxel - based combination regimen has been widely examined and its effectiveness demonstrated [ 11 - 15 ] . thus , valid comparison between chemotherapy regimens is not feasible in the current retrospective study . rather , the role of consolidation chemotherapy should be investigated in the future . because consolidation chemotherapy was rarely applied in the current study , it is still not clear whether further improvement of treatment outcomes is possible from consolidation chemotherapy . a multinational phase iii randomized trial that compares ccrt with or without consolidation chemotherapy in inoperable stage iii nsclc was initiated by the authors at smc . the interim analysis showed a median os of 20.7 and 21.1 months in the ccrt alone arm and consolidation arm , respectively ( p=0.49 ) . in addition , the corresponding median time to progression was 9.0 and 13.9 months , respectively ( p=0.19 ) . long - term results are expected to validate the feasibility and efficacy of consolidation chemotherapy . it should be noted that the current study has some potential limitations as a result of its retrospective nature , as well as the small number of patients and heterogeneous patient population . as recommended by the current nccn guideline , definitive bimodality therapy could be applied as a primary treatment in patients with n2-iiia nsclc that is unresectable or necessitates pneumonectomy , while trimodality could be considered for candidates that do not need pneumonectomy . additional efforts to avoid and minimize treatment break should be applied through a multidisciplinary team approach .

orthosiphon stamineus benth ( syn . : o. aristatus ( bl . ) miq . , misai kucing , leaves extracts have been used as traditional medicine and possess benefits such as antidiabetic , ability to increase plasma triglyceride and plasma hdl - cholesterol concentrations , anti - lithiatic and hypouricemic effects [ 4 , 5 ] , antifungal , and ability to treat kidney stone and urinary tract diseases [ 79 ] . hypertension has been reported to be associated with endothelium dysfunction in both human and animal studies . endothelium regulates vascular tone by releasing vasoconstrictors such as endothelins , prostanoids and oxygen reactive species , and vasodilators such as nitric oxide ( no ) , prostacyclin ( pgi2 ) , and endothelial hyperpolarizing factor ( edhf ) . these vasodilators were a great discovery by furchgott and zawadzki in 1980 , known as endothelium derived relaxing factors ( edrf ) . it has been reported by peach et al . that releases of edrf caused vasorelaxant effects of acetylcholine ( ach ) , which is dependent on the presence of the endothelial cells [ 11 , 13 ] . phenylephrine ( pe ) is a selective 1-adrenergic receptor agonist that increases arterial blood pressure by peripheral vasoconstriction . 1-adrenergic receptors which exist postsynaptically are g - protein - coupled receptors , and thus activation of cellular signaling is subsequent to the interaction with a g - protein . since pe is a selective 1-adrenergic receptor agonist and losartan is at1 receptor blocker , there is possible relationship between at1 and 1 receptors . in addition , crosstalk between at1 and 1 receptors in the smooth muscle of rabbit aorta is endothelium dependent . it has been reported that mrc a isolated from orthosiphon stamineus causes continuous decreases in systolic blood pressure ( sbp ) and heart rate ( hr ) after subcutaneous administration in conscious shr . the present study aimed to investigate the vascular reactivity of water extract ( wos ) and water : methanolic ( 1 : 1 ) extract ( wmos ) of orthosiphon stamineus benth and at1 receptors blocker in the mechanisms of antihypertensive effects mediated by 1-adrenergic receptor and prostacyclin ( pgi2 ) and endothelium - derived nitric oxide ( edno ) releases in the shr aortic rings . voucher specimen ( no . 11009 ) of the plant material was deposited at herbal room , school of pharmaceutical sciences , universiti sains malaysia ( usm ) . wmos was prepared by having dried and ground orthosiphon stamineus leaves extracted by a mixture of methanol : water ( 1 : 1 ) using a soxhlet extractor for a period of 12 hours , whereas preparation of wos involved hot maceration of the dried and ground orthosiphon stamineus leaves at 50c for 6 hours and was repeated thrice . each extract was bulked and concentrated in a rotary evaporator under vacuum and then freeze - dried and kept in a freezer until used . wos and wmos were freshly prepared in distilled water prior to the feeding of the animals . male spontaneously hypertensive rats ( shr , 230280 g ) were housed in individual cages with free access to foods and water and maintained at animal transit facility of school of pharmaceutical sciences , usm . all procedures involving animals were conducted according to the ethical guidelines by the animal ethics committee , usm . the animals were divided into four groups : ( 1 ) wos , 1000 mg / kg ; ( 2 ) wmos , 1000 mg / kg ; ( 3 ) losartan , 10 mg / kg ; and ( 4 ) control ( vehicle ) . all animals were given daily treatment orally for 14 days before being subjected to vascular reactivity studies . phenylephrine hydrochloride ( pe ) , acetylcholine ( ach ) , indomethacin , and n - nitro - l - arginine methyl ester ( l - name ) were purchased from sigma - aldrich , germany , while sodium chloride ( nacl ) , potassium chloride ( kcl ) , potassium dihydrogen phosphate ( kh2po4 ) , magnesium sulphate ( mgso47h2o ) , glucose , sodium hydrogen carbonate ( nahco3 ) , and calcium chloride dehydrate ( cacl2h2o ) were purchased from r&m chem . , uk . all drugs were freshly prepared in normal saline , except indomethacin in 0.5% ( w / v ) sodium carbonate , prior to use . the rat was anesthetized with sodium pentobarbital ( 60 mg / kg , i.p . ) . the thoracic aorta was carefully isolated , cleaned from the adherent fat and connective tissues , and cut into 35 mm rings . the aortic rings were then suspended horizontally in tissue chambers containing 10 ml of kreb 's solution ( mmol / l : nacl 118.6 , kcl 4.8 , cacl2 2.5 , mgso47h2o 1.2 , kh2po4 1.2 , nahco3 25.1 , and glucose 11.0 ) . the tissue - bath solution was bubbled incessantly with 95% o2 and 5% co2 ( carbogen ) at 37c . aortic rings were then allowed to equilibrate at an optimal tension of 1 g for 45 min . kreb 's solution was replaced every 15 min , and the tension was readjusted to 1 g when necessary . at the beginning of the experiment , the presence of intact endothelial cells was confirmed by precontracting the tissues with pe ( 1 m ) and followed by relaxation with ach ( 1 m ) . responses were recorded isometrically via a force transducer ( grass ft03d ) connected to a computerized data acquisition system ( powerlab ; adinstruments pty ltd . , the concentration - response curves for pe ( cumulative final chamber concentration of 10 to 10 m ) were recorded . the contraction effects of pe were recorded in two different preparations , intact and denude endothelium . denude endothelium of aortic rings was obtained by gently rubbing the intimal layer of the tissue with a blunt needle for a few times . the aortic rings were considered denuded when there were less than 10% relaxations to ach ( 1 m ) precontracted with pe ( 1 m ) whereas in order to obtain the concentration - response curves of relaxation , ach ( 10 to 10 m ) was added cumulatively to the chamber at the plateau of the pe ( 1 m ) precontracted aortic rings at 3-minute intervals . to further assess the involvement of edno and prostacyclin ( pgi2 ) releases , relaxations of aortic rings were performed in wos , wmos , and losartan groups preincubated for 30 minutes with l - name ( 100 m ) , a nonspecific no synthase inhibitor , and indomethacin ( 10 m ) , a nonselective cyclooxygenase inhibitor , respectively . pe - induced contraction and ach - induced relaxation were analysed using one - way anova followed by dunnett 's post hoc test , whereas the effects of ach - induced relaxation after preincubated by l - name and indomethacin were analysed using student 's t - test . emax , all analyses were using the computer software graphpad prism 5.0 for windows ( graphpad software inc . cumulative additions of pe ( 10 to 10 m ) produced a concentration - dependent contraction of aortic rings in all groups . in pe intact , wos , wmos , and losartan significantly decreased ( p < 0.05 ) the contractile responses as compared to control whereas , in pe denude endothelium , no significant changes were obtained ( figure 1 ) . maximal contractile responses ( emax ) in pe intact were significantly decreased in wos , wmos , and losartan ( 0.30 0.06 , 0.33 0.03 , 0.35 0.03 versus 0.90 0.10 ) . in contrast , the emax of wmos significantly enhanced the contraction responses in pe denude ( 0.57 0.02 versus 0.43 0.04 ) . the pd2 values from both pe intact and denude endothelium were unaltered as shown in table 1 . ach ( 10 to 10 m ) produced dose - dependent relaxation in all groups in aortic rings precontracted with pe ( 1 m ) . only losartan significantly enhanced ( p < 0.05 ) the relaxant effect of ach as compared to control ( rmax 111.20 4.08 versus 73.15 3.03 ) . both extract groups did not significantly alter the vasorelaxant effects of ach as shown in figure 2 and table 2 . to assess the contribution of edno , the aortic rings were preincubated with l - name ( 100 m ) , a no synthase inhibitor for 30 minutes . ach - induced relaxations in all groups were significantly inhibited ( p < 0.05 ) by l - name as shown in figure 3 . rmax and pd2 values were tabulated in table 2 . to investigate the role of prostacyclin ( pgi2 ) releases , the aortic rings were preincubated with indomethacin ( 10 m ) , a cox inhibitor for 30 minutes . indomethacin significantly reduced ( p < 0.05 ) the ach - induced relaxations in losartan and in contrast , significantly improved vasorelaxation in wmos ( figure 4 ) . rmax and pd2 values were tabulated in table 2 . to assess the role of intracellular and extracellular calcium mobilization , the aortic rings were incubated in ca - free medium containing 0.1 mm egta . under this condition , pe induced transient contraction mainly from sarcoplasmic reticulum . in endothelium - denuded aortic rings , a transient contractile response in ca - free medium the percentage contractile responses to pe were significantly reduced ( p < 0.05 ) in losartan ( 30.69 4.41% ) and wos ( 25.35 1.61% ) as compared to control ( 48.69 7.59% ) in response to pe in ca - free medium ( figure 5 ) . when the same procedure was repeated in normal ca2-containing medium which contained 2.5 mm cacl2 , the present study demonstrated that , in intact endothelium , the contractile response to phenylephrine ( pe ) , a selective agonist for a1-adrenergic receptor , was significantly lowered in shr treated with wos and wmos as compared to control . these results showed that 14-day oral treatment of wos and wmos affected the 1-adrenergic receptors activities in this preparation . the use of pe as a vasoconstrictor in the present study because the rat 's aorta has predominantly 1-postjunctional receptors . . demonstrated that suppression of constrictor responses to several agonists such as pe in the intact vascular endothelium may be due continuously basal release of endothelium - derived relaxing factor ( edrf ) from endothelial cells . as seen in the present study , wos and wmos inhibited the contraction induced by pe as comparable to losartan . there were studies found that possible crosstalk between at1 and 1-adrenoceptors existed [ 2123 ] . furthermore , maeso et al . reported that losartan reduced vasoconstrictor responses to pe in shr aortic rings via endogenous angiotensin ii ( angii ) acting on at1 receptors . activation of at1 receptors results in increasing systolic blood pressure ( sbp ) , blood vessels growth , and associated vascular smooth muscle cells ( vsmc ) apoptosis . blockade of at1 receptors which inhibit the effects of angii may promote good prognosis in pathological conditions such as hypertension and to inhibit vasoconstriction . hence , we may suggest that ( 1 ) orthosiphon stamineus leaves extracts exert their antihypertensive effects by blunting the increase of blood pressure in shr ; and ( 2 ) orthosiphon stamineus leaves extracts may play their role in reducing vasoconstriction similar as at1 receptor blocker . wos and wmos may possibly possess antihypertensive properties and exert similar effects through these interactions . it is likely that contribution by the variable edrf such as no , prostacyclin , and edhf caused vascular smooth muscle cells to relax . the necessary endothelial cells for the relaxation by acetylcholine ( ach ) to be occurred have been discovered since 1980 by furchgott and zawadzki . they demonstrated that loss of endothelium by rubbing the intimal surface of aorta caused no relaxation induced by ach . ach acts on muscarinic receptors of these cells thus stimulates substances that caused relaxation of vascular smooth muscle cells . in the present study , aortic rings from both wos and wmos showed essentially similar relaxant effects with control by dose - response manner to ach ( 10 to 10 m ) . it may speculate that both extracts given orally did not alter the endothelium of the rats ; thus the aortic rings isolated from these rats showed no effect to the ach - induced relaxation . it is plausible that blockade of at1 receptor further enhanced the relaxation caused by ach . furthermore , schiffrin and touyz demonstrated that losartan enhanced the endothelium - dependent relaxation to ach in shr aortic rings . in this preparation , the relaxation to ach was probably due to the production or release of edrf and the endothelial vasorelaxant factors derived from cyclooxygenase ( cox ) pathways ( prostacyclin pgi2 released from endothelial cells ) . this similar effect has been reported by luscher and vanhoutte . however , wmos improved vasorelaxation to ach after blockade of cox pathways . in this case , there was plausibility because the vasodilator pgi2 was continuously released as indicated by its tonic effects on platelet cyclic adenosine monophosphate ( camp ) . in contrast , losartan significantly reduced the ach - induced relaxations , which may be due to the attenuation of pgi2 production which was compensated for by the enhanced release of another vasodilator , for example , nitric oxide ( no ) . in this point of view , we might suggest that wmos and blockade of at1 receptors modulate the derived endothelial vasorelaxant factors such as pgi2 from cox pathways . no to play a vital role in the maintenance of vascular tone . in order to assess the contribution of no releases in the vasorelaxant effects elicited by orthosiphon stamineus leaves extracts , we preincubated the rat aortic rings with l - name ( 100 m ) , no synthase inhibitor . our study showed that the ach - induced vasorelaxation in all treatment groups reduced significantly after inhibition of no synthase pathways . from the present data , it could be clearly proven that no synthase pathways were involved in the vasorelaxation in the shr . in view of the present study , it is plausible that the vasorelaxant activities produced by orthosiphon stamineus extracts may take place in the vascular smooth muscle cells . to investigate the effects of both extracts and losartan on the role intracellular ca on the contractility of the vascular smooth muscle cells of the aortic rings , significantly reduced contraction response to pe in denude aortic rings observed in the wos and losartan were possibly due to inhibition of intracellular ca release from the sarcoplasmic reticulum at the level of vascular smooth muscle cells . decreased intracellular ca concentration and increased myosin light chain phosphatase activity may had caused the smooth muscle to undergo weaker vascular contractility . also , inhibition of receptor- and voltage - operated ca channels in the plasma membrane reduced ca influx may contribute as well . in conclusion , our studies showed that water extract ( wos ) and water : methanolic ( 1 : 1 ) extract ( wmos ) of orthosiphon stamineus benth leaves promote antihypertensive effects by reducing vasoconstriction through the alteration of 1-adrenergic and at1 receptors activities . vasorelaxant effects of both wos and wmos may possibly involve mainly the release of edno . in wos , pgi2 releases might not be participated in the ach - induced vasorelaxation . however in wmos , enhancement of vasorelaxation might be due to the fact that vasodilator pgi2 is continuously released as indicated by its tonic effects on platelet camp . in addition , wos inhibited the contraction of aortic rings induced by pe , implying that wos inhibits the release of intracellular ca and/or blocks rocc .

the melany cohort has been conducted at the israel defense forces staff periodic examination center ( spec ) , to which all career service personnel aged older than 25 years are referred every 3 to 5 years for a routine health examination and screening tests , as described previously ( 22,23 ) . at each visit to the spec , participants completed a detailed questionnaire assessing demographic , nutritional , lifestyle , and medical factors . height and weight were measured , and a physician at the center performed a complete physical examination . primary care for all israel defense forces personnel between scheduled visits to the center is obtained at designated military clinics , and all medical information is recorded in the same central database , thereby facilitating ongoing , tight , and uniform follow - up . this study assessed 37,418 men who had documented wbc counts within the normal range ( 3,00012,000 cells / mm ) at their first visit to the spec between the years 1995 and 2010 . the analysis excluded 1 ) men with pre - existing type 1 or type 2 diabetes , men with fasting plasma glucose ( fpg ) level 100 mg / dl at the first visit ( n = 3,638 ) , or men withnewly diagnosed diabetes ( fpg 126 mg / dl , n = 602 ) ; 2 ) men with a follow - up shorter than 2 years ( n = 8,001 ) ; or 3 ) men missing fasting glucose levels or wbc count ( n = 280 ) . the institutional review board of the israel defense forces medical corps approved this study on the basis of strict maintenance of participants anonymity during database analyses . participants aged 25 to 45 years were followed up prospectively from their first visit to the spec until retirement from military service . follow - up ended at the time of diabetes diagnosis , death , retirement from military service , or 8 march 2011 , whichever came first . mean total follow - up was 7.71 3.83 years , corresponding to 85 and 67% completing at least 3 and 5 years of follow - up , respectively . all participants were censored at the end of their follow - up period ( based on the above criteria for end of follow - up ) ; thus , none of the study participants were lost to follow - up . of note , follow - up duration for subjects who did and did not develop diabetes was similar ( 7.72 2.84 and 7.44 3.84 years , respectively , p = 0.13 ) , and baseline wbc levels did not affect the number of visits to the spec . the diagnosis of 447 incident cases of diabetes was based on the american diabetes association criteria by documenting two fpg levels of 126 mg / dl ( 7.0 mmol / l ) or a glucose level 126 mg / dl 2 h after ingestion of 75 g of glucose . the diagnosis was made after the abnormal screening result at each visit to the spec in 198 subjects ( 44.3% ) or between visits by the participants israeli defense force primary care physician , followed by confirmation by a military physicians committee , in 249 ( 55.7% ) . all laboratory studies were performed on fresh samples in an international organization for standardization ( iso)-9002 quality - assured core facility laboratory . the cohort was divided into wbc quintiles , and their baseline characteristics are presented in table 1 . ( baseline characteristics of patients who developed diabetes vs. those who did not are detailed in supplementary table 1 . ) the medians of the quintiles were fit as continuous variables to estimate the trend of variables across quintiles in a linear regression model ( adjusted r = 0.99 , b = 900 cells / mm per quintile , p = 0.001 ) . cox proportional hazard models were used to estimate the hazard ratios ( hr ) and 95% cis for developing diabetes . we gradually added to the age - adjusted model ( model 1 ) additional parameters known as classical risk factors for diabetes ( table 2 ) . in model 2 , the following categorical variables were added to model 2 : smoking status ( current smoker , former smoker , never smoked ) , family history of diabetes ( yes or no ) , and physical activity ( not active , < 150 min / week , 150 min / week ) . in model 4 , serum triglyceride levels ( quintiles ) and an additional multivariate model was conducted analyzing wbc count as a continuous variable . of note , the presence or absence of a family history of diabetes baseline characteristics of population cohort hr for developing diabetes in normoglycemic patients across wbc quintiles log - minus - log plots for each variable were inspected to verify the assumption of proportionality of the hazards . the maximal r recorded was 0.357 ( triglyceride and bmi ) . omnibus tests of model coefficients were used to assess the relative contribution of the various variables to the model . to evaluate the power of the models to discriminate events from nonevents we calculated the area under the receiver operating characteristic ( roc ) curve for each of the variables as well as in a multivariate model ( c statistic ) . to study potential threshold in wbc count for diabetes prediction , a restricted cubic spline ( using r software ) and a decision tree procedure with chaid ( automatic interaction detection ) method were used . values are reported as mean standard deviation ( sd ) , unless mentioned otherwise . the melany cohort has been conducted at the israel defense forces staff periodic examination center ( spec ) , to which all career service personnel aged older than 25 years are referred every 3 to 5 years for a routine health examination and screening tests , as described previously ( 22,23 ) . at each visit to the spec , participants completed a detailed questionnaire assessing demographic , nutritional , lifestyle , and medical factors . height and weight were measured , and a physician at the center performed a complete physical examination . primary care for all israel defense forces personnel between scheduled visits to the center is obtained at designated military clinics , and all medical information is recorded in the same central database , thereby facilitating ongoing , tight , and uniform follow - up . this study assessed 37,418 men who had documented wbc counts within the normal range ( 3,00012,000 cells / mm ) at their first visit to the spec between the years 1995 and 2010 . the analysis excluded 1 ) men with pre - existing type 1 or type 2 diabetes , men with fasting plasma glucose ( fpg ) level 100 mg / dl at the first visit ( n = 3,638 ) , or men withnewly diagnosed diabetes ( fpg 126 mg / dl , n = 602 ) ; 2 ) men with a follow - up shorter than 2 years ( n = 8,001 ) ; or 3 ) men missing fasting glucose levels or wbc count ( n = 280 ) . the institutional review board of the israel defense forces medical corps approved this study on the basis of strict maintenance of participants anonymity during database analyses . participants aged 25 to 45 years were followed up prospectively from their first visit to the spec until retirement from military service . follow - up ended at the time of diabetes diagnosis , death , retirement from military service , or 8 march 2011 , whichever came first . mean total follow - up was 7.71 3.83 years , corresponding to 85 and 67% completing at least 3 and 5 years of follow - up , respectively . all participants were censored at the end of their follow - up period ( based on the above criteria for end of follow - up ) ; thus , none of the study participants were lost to follow - up . of note , follow - up duration for subjects who did and did not develop diabetes was similar ( 7.72 2.84 and 7.44 3.84 years , respectively , p = 0.13 ) , and baseline wbc levels did not affect the number of visits to the spec . the diagnosis of 447 incident cases of diabetes was based on the american diabetes association criteria by documenting two fpg levels of 126 mg / dl ( 7.0 mmol / l ) or a glucose level 126 mg / dl 2 h after ingestion of 75 g of glucose . the diagnosis was made after the abnormal screening result at each visit to the spec in 198 subjects ( 44.3% ) or between visits by the participants israeli defense force primary care physician , followed by confirmation by a military physicians committee , in 249 ( 55.7% ) . all laboratory studies were performed on fresh samples in an international organization for standardization ( iso)-9002 quality - assured core facility laboratory . the cohort was divided into wbc quintiles , and their baseline characteristics are presented in table 1 . ( baseline characteristics of patients who developed diabetes vs. those who did not are detailed in supplementary table 1 . ) the medians of the quintiles were fit as continuous variables to estimate the trend of variables across quintiles in a linear regression model ( adjusted r = 0.99 , b = 900 cells / mm per quintile , p = 0.001 ) . cox proportional hazard models were used to estimate the hazard ratios ( hr ) and 95% cis for developing diabetes . we gradually added to the age - adjusted model ( model 1 ) additional parameters known as classical risk factors for diabetes ( table 2 ) . in model 2 , the following categorical variables were added to model 2 : smoking status ( current smoker , former smoker , never smoked ) , family history of diabetes ( yes or no ) , and physical activity ( not active , < 150 min / week , 150 min / week ) . in model 4 , serum triglyceride levels ( quintiles ) and fasting glucose level ( as continuous variables ) were added to the model . an additional multivariate model was conducted analyzing wbc count as a continuous variable . of note , the presence or absence of a family history of diabetes baseline characteristics of population cohort hr for developing diabetes in normoglycemic patients across wbc quintiles log - minus - log plots for each variable were inspected to verify the assumption of proportionality of the hazards . omnibus tests of model coefficients were used to assess the relative contribution of the various variables to the model . to evaluate the power of the models to discriminate events from nonevents we calculated the area under the receiver operating characteristic ( roc ) curve for each of the variables as well as in a multivariate model ( c statistic ) . to study potential threshold in wbc count for diabetes prediction , a restricted cubic spline ( using r software ) and a decision tree procedure with chaid ( automatic interaction detection ) method were used . values are reported as mean standard deviation ( sd ) , unless mentioned otherwise . data of 24,897 apparently healthy young normoglycemic men who were followed up as part of the melany cohort met the inclusion criteria . mean wbc count was 6,620 1,480 cells / mm ( range 3,00012,000 ) with an average increment of 900 cells / mm between consequent quintiles . wbc level was directly correlated with bmi , systolic and diastolic blood pressure , triglyceride level , ldl - cholesterol , and rates of current smokers . physical activity and hdl cholesterol were inversely correlated with wbc level ( p < 0.0001 for trend ) . during 185,354 person - years of follow - up the incidence of type 2 diabetes increased linearly across quintiles of wbc , with 62 new cases diagnosed in the lowest quintile ( q1 ; wbc of 3,0005,400 cells / mm ) and 124 new cases diagnosed in q5 ( wbc > 7,810 ) . in model 1 , adjusted for age , the risk for type 2 diabetes was 2-fold higher in q4 ( wbc 6,9107,800 cells / mm ) and 2.44-fold higher in q5 compared with q1 ( hr 2.44 [ 95% ci 1.793.31 ] ) . further adjustment for bmi ( model 2 , table 2 ) significantly attenuated the risk for diabetes associated with wbc levels . nevertheless , even in the multivariate model adjusted for age , bmi , family history of diabetes , physical activity , triglyceride level , and fasting glucose , wbc count remained an independent risk factor of incident diabetes at levels > 6,910 cells / mm ( model 4 , table 2 ) , with a hr in q4 versus q1 of 1.48 ( 1.032.14 ; p = 0.011 for trend ) . when wbc count was modeled as a continuous variable , an increment of 1,000 cells / mm in wbc count was associated with a 7.6% increase in the risk for developing diabetes independently of age , bmi , family history of diabetes , physical activity , triglyceride level , and fasting glucose level ( hr 1.076 [ 1.0011.157 ] , p = 0.046 ) . to better assess the interrelation between obesity and wbc , we next studied the joint effect of bmi and wbc levels in predicting the risk for developing type 2 diabetes ( fig . 1a ) . in a multivariate analysis controlled for age , family history of diabetes , physical activity , triglyceride level , and fpg , obese men ( bmi 30 kg / m ) with elevated wbc count within the normal range ( 7,81012,000 cells / mm ) had a more than sixfold increase in the risk for type 2 diabetes ( hr 6.1 [ 95% ci 3.819.78 ] ) compared with the reference group . of interest , a strong attenuation in the risk for type 2 diabetes attributed to overweight and obesity was observed among individuals with low - normal wbc ( < 5,400 cells / mm ) , reaching an hr of 2.63 ( 1.195.88 ) for obese men and a nonsignificant hr of 1.40 ( 0.82.44 ) for overweight men . taken together , these observations suggest that , in the presence of a low - normal wbc count , overweight and obese men are relatively protected from the development of type 2 diabetes compared with overweight or obese individuals with higher wbc count . risk factor for diabetes and wbc count in young normoglycemic patients . the dark bars indicate a significant change compared with the reference group . a : joint effect of bmi and wbc count and the risk for incident diabetes . data are adjusted for age , family history of diabetes , activity status , smoking status , blood pressure , triglycerides level , and fasting glucose . the reference group is in the lower wbc quintile at the lower ( lean ) bmi tertile . b : joint effect of plasma triglycerides ( tg ) level and wbc count and the risk for diabetes . data are adjusted for age , bmi , family history of diabetes , activity status , smoking status , and fasting glucose . reference group is the lower wbc quintile at a normal ( < 150 mg / dl ) tg level . c : joint effect of family history of diabetes , wbc count , and the risk for diabetes . data are adjusted for age , bmi of diabetes , activity status , smoking status , triglycerides level , and fasting glucose . similar to the observation with bmi , a relatively low wbc level ( < 5,400 cells / mm ) was associated with complete attenuation of diabetes risk attributed to an elevated triglyceride level or to a positive family history of diabetes . we next assessed for a potential threshold within the normal range of the wbc count , above which diabetes risk increases significantly . although a decision tree procedure ( with chaid ) suggested a wbc value of 7,230 cells / mm as a potential cut point , no evidence for a nonlinear component of wbc was observed using restricted cubic splines ( data not shown ) . we therefore performed the cox regression multivariate models using a moving cutoff value for wbc in increments as low as 100 cells / mm ( supplementary table 4 ) . a clear threshold could not be delineated ; however , the highest hr ( 1.438 ) with the lowest p value ( 0.002 ) for diabetes incidence was observed when the value of 7,100 cells / mm was used . of note , wbc > 7,100 cells / mm corresponds exactly to levels observed among the upper one - third of the study population . to assess the differential contribution of the various risk factors to the prediction of diabetes , we next calculated the roc curves for wbc count and for other , well - validated diabetes risk factors . surprisingly , the auc of the wbc count roc curve was not statistically different than that of fasting glucose ( 0.59 [ 95% ci 0.550.61 ] vs. 0.58 [ 0.550.61 ] , respectively ) . bmi was the single most powerful predictor for diabetes ( auc 0.670 [ 0.6430.696 ] ) , followed by triglyceride level ( 0.63 [ 0.600.66 ] ) , wbc count , and fasting glucose . the addition of all parameters included in model 4 resulted in an auc of 0.674 ( 0.6430.705 ) without wbc and 0.676 ( 0.6450.707 ) with wbc . the change in 2 log likelihood with addition of each of the variables to the cox regression model ( omnibus test of model coefficients ) revealed that bmi and age had the highest contribution to the model , followed by wbc count , family history of diabetes , and triglyceride level ( supplementary table 2 ) . as detailed above , the upper one - third of the wbc distribution ( corresponding to wbc levels > 7,100 cells / mm ) was suggested as a potential threshold for increased diabetes risk . indeed , a significant contribution to the full multivariate model was observed when tertiles of wbc ( < 5,900 , 5,9107,100 , or 7,110 cells / mm ) were used ( p= 0.004 omnibus test ) . of the 24,897 subjects included in this study , 7,939 were of african origin , exhibiting a lower mean of wbc count ( 5,510 1,440 cells / mm , p < 0.001 ) than participants from non - african descent . when wbc count was modeled as a continuous variable , a significant 11.8% increase in diabetes risk was observed for every increment of 1,000 cells / mm in this population ( hr 1.118 [ 95%ci 1.011.25 ] , p = 0.05 ) . these data indicate that wbc count may be considered as a risk factor even among subjects with a genetic tendency for a lower range of wbc count ( further detailed in supplementary table 3 ) . data of 24,897 apparently healthy young normoglycemic men who were followed up as part of the melany cohort met the inclusion criteria . mean wbc count was 6,620 1,480 cells / mm ( range 3,00012,000 ) with an average increment of 900 cells / mm between consequent quintiles . wbc level was directly correlated with bmi , systolic and diastolic blood pressure , triglyceride level , ldl - cholesterol , and rates of current smokers . physical activity and hdl cholesterol were inversely correlated with wbc level ( p < 0.0001 for trend ) . during 185,354 person - years of follow - up , 447 new cases of diabetes were diagnosed among young normoglycemic men . the incidence of type 2 diabetes increased linearly across quintiles of wbc , with 62 new cases diagnosed in the lowest quintile ( q1 ; wbc of 3,0005,400 cells / mm ) and 124 new cases diagnosed in q5 ( wbc > 7,810 ) . in model 1 , adjusted for age , the risk for type 2 diabetes was 2-fold higher in q4 ( wbc 6,9107,800 cells / mm ) and 2.44-fold higher in q5 compared with q1 ( hr 2.44 [ 95% ci 1.793.31 ] ) . further adjustment for bmi ( model 2 , table 2 ) significantly attenuated the risk for diabetes associated with wbc levels . nevertheless , even in the multivariate model adjusted for age , bmi , family history of diabetes , physical activity , triglyceride level , and fasting glucose , wbc count remained an independent risk factor of incident diabetes at levels > 6,910 cells / mm ( model 4 , table 2 ) , with a hr in q4 versus q1 of 1.48 ( 1.032.14 ; p = 0.011 for trend ) . when wbc count was modeled as a continuous variable , an increment of 1,000 cells / mm in wbc count was associated with a 7.6% increase in the risk for developing diabetes independently of age , bmi , family history of diabetes , physical activity , triglyceride level , and fasting glucose level ( hr 1.076 [ 1.0011.157 ] , p = 0.046 ) . to better assess the interrelation between obesity and wbc , we next studied the joint effect of bmi and wbc levels in predicting the risk for developing type 2 diabetes ( fig . 1a ) . in a multivariate analysis controlled for age , family history of diabetes , physical activity , triglyceride level , and fpg , obese men ( bmi 30 kg / m ) with elevated wbc count within the normal range ( 7,81012,000 cells / mm ) had a more than sixfold increase in the risk for type 2 diabetes ( hr 6.1 [ 95% ci 3.819.78 ] ) compared with the reference group . of interest , a strong attenuation in the risk for type 2 diabetes attributed to overweight and obesity was observed among individuals with low - normal wbc ( < 5,400 cells / mm ) , reaching an hr of 2.63 ( 1.195.88 ) for obese men and a nonsignificant hr of 1.40 ( 0.82.44 ) for overweight men . taken together , these observations suggest that , in the presence of a low - normal wbc count , overweight and obese men are relatively protected from the development of type 2 diabetes compared with overweight or obese individuals with higher wbc count . risk factor for diabetes and wbc count in young normoglycemic patients . the dark bars indicate a significant change compared with the reference group . a : joint effect of bmi and wbc count and the risk for incident diabetes . data are adjusted for age , family history of diabetes , activity status , smoking status , blood pressure , triglycerides level , and fasting glucose . the reference group is in the lower wbc quintile at the lower ( lean ) bmi tertile . b : joint effect of plasma triglycerides ( tg ) level and wbc count and the risk for diabetes . data are adjusted for age , bmi , family history of diabetes , activity status , smoking status , and fasting glucose . reference group is the lower wbc quintile at a normal ( < 150 mg / dl ) tg level . c : joint effect of family history of diabetes , wbc count , and the risk for diabetes . data are adjusted for age , bmi of diabetes , activity status , smoking status , triglycerides level , and fasting glucose . similar to the observation with bmi , a relatively low wbc level ( < 5,400 cells / mm ) was associated with complete attenuation of diabetes risk attributed to an elevated triglyceride level or to a positive family history of diabetes . we next assessed for a potential threshold within the normal range of the wbc count , above which diabetes risk increases significantly . although a decision tree procedure ( with chaid ) suggested a wbc value of 7,230 cells / mm as a potential cut point , no evidence for a nonlinear component of wbc was observed using restricted cubic splines ( data not shown ) . we therefore performed the cox regression multivariate models using a moving cutoff value for wbc in increments as low as 100 cells / mm ( supplementary table 4 ) . a clear threshold could not be delineated ; however , the highest hr ( 1.438 ) with the lowest p value ( 0.002 ) for diabetes incidence was observed when the value of 7,100 cells / mm was used . of note , wbc > 7,100 cells / mm corresponds exactly to levels observed among the upper one - third of the study population . to assess the differential contribution of the various risk factors to the prediction of diabetes , we next calculated the roc curves for wbc count and for other , well - validated diabetes risk factors . surprisingly , the auc of the wbc count roc curve was not statistically different than that of fasting glucose ( 0.59 [ 95% ci 0.550.61 ] vs. 0.58 [ 0.550.61 ] , respectively ) . bmi was the single most powerful predictor for diabetes ( auc 0.670 [ 0.6430.696 ] ) , followed by triglyceride level ( 0.63 [ 0.600.66 ] ) , wbc count , and fasting glucose . the addition of all parameters included in model 4 resulted in an auc of 0.674 ( 0.6430.705 ) without wbc and 0.676 ( 0.6450.707 ) with wbc . the change in 2 log likelihood with addition of each of the variables to the cox regression model ( omnibus test of model coefficients ) revealed that bmi and age had the highest contribution to the model , followed by wbc count , family history of diabetes , and triglyceride level ( supplementary table 2 ) . as detailed above , the upper one - third of the wbc distribution ( corresponding to wbc levels > 7,100 cells / mm ) was suggested as a potential threshold for increased diabetes risk . indeed , a significant contribution to the full multivariate model was observed when tertiles of wbc ( < 5,900 , 5,9107,100 , or 7,110 cells / mm ) were used ( p= 0.004 omnibus test ) . of the 24,897 subjects included in this study , 7,939 were of african origin , exhibiting a lower mean of wbc count ( 5,510 1,440 cells / mm , p < 0.001 ) than participants from non - african descent . when wbc count was modeled as a continuous variable , a significant 11.8% increase in diabetes risk was observed for every increment of 1,000 cells / mm in this population ( hr 1.118 [ 95%ci 1.011.25 ] , p = 0.05 ) . these data indicate that wbc count may be considered as a risk factor even among subjects with a genetic tendency for a lower range of wbc count ( further detailed in supplementary table 3 ) . in this prospective , large - scale study , we demonstrate that a single measurement of wbc in healthy , normoglycemic young men may predict diabetes incidence independently from the other traditional risk factors for diabetes such as bmi , fasting glucose and triglyceride levels , and family history . we report that for every increase in 1,000 cells / mm within the normal range of the wbc count , the risk for diabetes increases by 7.6% , and levels of wbc already as low as 6,900 cells / mm and above are associated with an obesity - independent 50% increase in the risk for diabetes . in addition , overweight and obese subjects with a relatively low wbc count seem to have a significantly lower risk for diabetes than those with higher levels of leukocytes , which may help in better characterizing obese individuals who are relatively protected from the metabolic derangements frequently associated with obesity . given the important role that has been suggested for chronic inflammation in obese states and its causal role in mediating insulin resistance in peripheral tissues ( 24 ) , incorporating novel inflammatory markers into diabetes risk prediction models has recently raised interest ( 2527 ) . assessment of the inflammatory burden associated with obesity may contribute to the interpretation of commonly used clinical measurements of adiposity , such as bmi , waist circumference , and hip - to - thigh ratio , in determining the inflammatory consequences of increased adiposity and the risk for diabetes . indeed , many studies have demonstrated a positive association between increased levels of inflammatory markers , such as wbc count , crp , and inflammatory cytokines , and diabetes incidence , although only a few have tested this prospectively in large , population - based studies ( for a systematic review and meta - analysis , please see reference 18 ) . previous studies have been inconsistent in whether wbc or other inflammatory markers contribute to diabetes prediction models independent of obesity ( 8,9,1921 ) or whether these markers simply reflect adipose tissue mass ( 11,13,17,19 ) . the current study , to the best of our knowledge , is the largest cohort to address the potential role of wbc as a common , widely available , and simple method to measure inflammatory biomarker for diabetes prediction in young adults . in the population studied , elevated wbc count was not associated with increased diabetes risk in lean , normoglycemic men , a subgroup at a very low risk for diabetes ( total incidence of 0.90% for q1 to 0.99% for q5 during 7.5 years for individuals with bmi < 25 kg / m , fig . however , wbc count was able to differentiate between subgroups of overweight and obese young men who are at a relatively low risk ( or even without an excess risk ) for diabetes and those who progressed to overt diabetes at significantly higher rates . a potential explanation for the lack of predictive value of wbc count for diabetes incidence among men who are not overweight or obese men is that wbc count is a cumulative estimation of the whole - body inflammatory burden , which reflects chronic as well as acute inflammatory processes . adipose tissue inflammation has mainly been attributed to inflammatory processes observed in visceral and/or ectopic fat depositions , compartments whose relative mass is highly increased in obesity ( 28 ) . therefore , young , healthy , lean men , who have less visceral and ectopic fat mass than obese subjects , will be less likely to exhibit significant adipose tissue inflammation ( 29 ) . in this setting , and in the absence of other recognized chronic inflammatory processes , a relatively elevated wbc count may represent more acute , intercurrent processes that do not seem to be associated with a higher rate of diabetes incidence . ( 30 ) demonstrated that visceral but not subcutaneous adipose tissue mass is associated with insulin resistance independent of bmi . their results , as well as others ( 31,32 ) , emphasize that the body distribution of fat , rather than cumulative fat mass as denoted by bmi , is a principle determinant of inflammation . in addition , the results of the current study suggest that chronic , mild inflammation is an important and perhaps a necessary step in the progression of young adults to diabetes , because subjects with low wbc counts do not exhibit an excess risk for incident diabetes , even in the presence of excess weight , an elevated triglyceride level , or a positive family history of diabetes . first , the melany cohort may be considered representative of a unique group of healthy young men . however , the characteristics of the population are strikingly similar to those of cohorts in published studies of young men from various industrialized countries ( 3335 ) . in addition , the relatively homogeneous environment to which participants in our study were exposed might reduce the effect of unknown confounders . second , measurements of waist circumference , circulating insulin , and c - peptide were not obtained in this study . although this did not compromise the outcome definition , it limited our ability to assess the role of insulin resistance in the association between wbc count and diabetes . finally , we did not measure glycosylated hemoglobin levels or perform glucose - tolerance tests . the current definition of normal fpg levels resulted in a substantial increase in the overlap with normal glucose tolerance , as defined by glucose - tolerance testing ( 36 ) ; however , we may have missed men with normal fpg levels who were already glucose intolerant at enrollment . to limit this possibility , we confirmed our results by performing a secondary analysis in which a 2-year lag between enrollment and outcome was used . another point worth discussing is the availably of only one wbc count value at baseline , which may not accurately represent the true inflammatory burden in the presence of acute illnesses or stresses . these potential false - positive results , however , could have only underestimated the association of wbc count with diabetes incidence . the strengths of the melany study include the detailed , uniform , and systematic follow - up and outcome definition , the use of measured ( rather than reported ) values for the bmi , the availability of reliable determinations of glucose levels in fresh venous blood , and the direct measurements of lipids . the identification of a high - normal wbc count , measured in an outpatient screening setting , as a risk factor for type 2 diabetes in addition to the other known risk factors may elaborate the interpretation of this commonly used laboratory test by primary care providers , increasing their attention to increased diabetes risk among young adults , who are usually not frequently screened for this condition . identifying individuals at higher risk for lifestyle and pharmacological approaches have both been reported to be efficient in delaying the onset of diabetes in selected groups at high risk for the disease ( 3740 ) . of specific interest is the observation that specific anti - inflammatory treatments for rheumatoid arthritis patients also resulted in a decreased incidence of diabetes ( 6 ) . targeting the obesity - related chronic inflammatory response to prevent the metabolic derangements associated with increased fat deposition may become an attractive approach for diabetes prevention once additional , more specific anti - inflammatory treatments with more tolerated side effects become available .

transradial angiography ( tra ) and transradial coronary intervention ( tri ) have become popular approaches because of several advantages , such as fewer bleeding complications [ 13 ] . however , use of tra and tri is limited for patients with a narrow radial artery , being associated with the risk of artery spasm , occlusion or pain . indeed , the 6 fr sheath size is too big for some patients . the outer diameter of a 5 fr sheath may also be too wide because radial artery studies demonstrate that a 4 fr sheath is optimal for the majority of patients [ 4 , 5 ] . glidesheath slender ( gss , terumo , japan ) is compatible with a 5 fr guiding catheter and it was designed solely for tr access . all the 5fr guiding catheters , which we can use in japan 2015 , are compatible with the 5fr gss that was already checked in vitro . it has a thinner wall , which reduces the outer diameter by 1 fr , while the diameter of the inner lumen is unchanged ( fig . 1 ) . aminian et al . reported the safety and feasibility of the 6 fr gss . in this study , initial experience with the 5 fr gss ( 4 fr outer diameter ) was evaluated in patients undergoing tra and tri , focusing on safety and feasibility.fig . 1the novel introducer sheath of the 5 fr glidesheath slender has an inner lumen the same size as a conventional 5 fr sheath , combined with an outer diameter similar to that of a conventional 4 fr sheath . the artery access size is 1 fr lower than that of a conventional 5 fr sheath the novel introducer sheath of the 5 fr glidesheath slender has an inner lumen the same size as a conventional 5 fr sheath , combined with an outer diameter similar to that of a conventional 4 fr sheath . the artery access size is 1 fr lower than that of a conventional 5 fr sheath tra or tri using the gss 5 fr was performed in tergooi hospital , the kingdom of the netherlands , on two consecutive days in january 2015 . 2 ) was suspected or confirmed ischemic heart disease requiring coronary angiography or percutaneous coronary intervention ( pci ) via the radial artery . ad hoc pci was planned for all patients , and preoperative administration of dual antiplatelet drugs was mandatory . exclusion criteria were as follows : no indication for coronary angiography or pci , less than 21 years old , females with the possibility of pregnancy , unable to obtain written informed consent , and patients whom the physician deemed to be unsuitable for this study.fig . , common color of 5fr sheath is gray ; the color of the 5fr gss is red which is for common 4fr sheath . because outer diameter of the 5fr gss is the same as that of conventional 4fr sheath 5fr gc was inserted to the 5fr gss via right radial approach however , common color of 5fr sheath is gray ; the color of the 5fr gss is red which is for common 4fr sheath . because outer diameter of the 5fr gss is the same as that of conventional 4fr sheath only procedures using the 5 fr gss were included . the approach was via either the right or left radial artery because the gss is approved exclusively for radial artery access . after local anesthesia , puncture with 21-gauge needles was performed , the guide wire was inserted , and the 5 fr gss was placed inside the radial artery . all patients received 5000 iu of heparin , irrespective of the scheduled procedure . the decision to perform ad hoc pci after tra was made by each physician . following procedure completion , the 5 fr gss was immediately removed , and hemostasis was performed with either tr - band ( terumo , japan ) or a bandage and small pillow , as selected by the physician . tr - band was removed 45 h after confirmation of bleeding cessation , while bandages were removed after the overnight hospital stay . the primary endpoint was radial artery patency after removal of hemostasis devices , and pulsation was compared with that before the procedure . for cases with no pulsation or a low level of pulsation , radial artery patency was determined with the pulse doppler method . secondary endpoints were : radial artery spasm , hematoma , major bleeding complication , functional disorder including hand ischemia due to radial artery dysfunction , pain score , and major adverse cardiac and cerebrovascular events ( macce ) related to the gss . radial spasm was defined clinically as significant limitation of the catheter movement felt by the operator with severe pain in the forearm of the patient . macce was defined as : all - cause mortality , cardiac death , myocardial infarction , cerebral bleeding , cerebral infarction , or acute revascularization . pain was assessed after sheath removal using the visual analog scale ( vas ) , with scores ranging from 0 to 10 cm . large hematoma was defined as more than 5 cm or equal to 5 cm in diameter , and small hematoma as less than 5 cm . major bleeding was defined as fatal bleeding , and/or symptomatic bleeding in a critical area or organ , and/or bleeding accompanied by either a decline in hemoglobin levels of greater than or equal to 20 g l ( 1.24 mmol l ) or the need for a transfusion of greater than or equal to 2 units of whole blood or red blood cells . a total of 21 patients were included , of whom 85.7 % were male . ( mean sd ) years , and the mean body mass index was 26.6 3.8 the mean number of previous attempts at radial artery access was 0.85 , and the maximum was six.table 1patients characteristics ( n = 21)age ( years)61.5 13.6 ( 3591)male gender ( % ) 18 ( 85.7)height ( cm)174 9.1weight ( kg)64.0 14.5bmi ( kg / m)26.6 3.8coronary risk factor , n ( % ) hypertension11 ( 52.4 ) dyslipidemia9 ( 42.8 ) diabetes6 ( 28.6 ) insulin treated2 ( 9.2 ) current smoker10 ( 47.6)number of previous radial access , n ( % ) 010 ( 47.6 ) 18 ( 38.1 ) 22 ( 9.5 ) 61 ( 7.8)procedure ( % ) angiography10 ( 47.6 ) ad hoc pci11 ( 52.4)procedure time ( min)24.1 9.2 bmi body mass index , pci percutaneous coronary intervention patients characteristics ( n = 21 ) bmi body mass index , pci percutaneous coronary intervention ad hoc pci was performed after tra in 11 patients , while the other 10 patients underwent tra only . the mean procedure time was 24.1 9.2 min the results of primary and secondary endpoints are shown in table 2 . no cases of radial artery occlusion were observed . in one patient who had undergone radial access six times previously , pulsation was very weak from the beginning of the procedure , and no change was detected after tr - band removal ; as a result , patency was assessed with the pulse doppler method.table 2results of the primary and secondary endpointspatency after removal of hemostasis devices ( % ) patency21(100.0 ) radial artery occlusion0spasm0major and minor hematoma0major bleeding0functional disorder0pain score0.57 ( 0.03.0)macce0macce : all - cause mortality , cardiac death , myocardial infarction , cerebral bleeding , cerebral infarction , or acute revascularization related to the glidesheath slender results of the primary and secondary endpoints macce : all - cause mortality , cardiac death , myocardial infarction , cerebral bleeding , cerebral infarction , or acute revascularization related to the glidesheath slender no cases of radial artery occlusion or spasm , and no bleeding complications were reported in this study . vas scores were very low , indicating that the sheath had a low propensity to cause pain . it should be noted that neither radial artery occlusion nor spasm occurred in a patient who had undergone tr access six times previously . the outer lumen of the 5 fr gss is the same as that of a conventional 4 fr sheath ; however , 5 fr catheters were used in this study , and the results are similar to those observed with 4 fr conventional sheaths . nevertheless , it was a concern that the thinner wall of the gss may be prone to kinking or fracture , but no such phenomenon occurred . it is well known that the use of a sheath with a larger diameter than that of the radial artery causes injury . reducing the sheath size from 5 fr to 4 fr is associated with safer tr access , with the rate of vascular access site complications reduced by up to 5 and 15 % in japanese males and females , respectively [ 4 , 5 ] . it is expected that a similar level of risk reduction may be achieved with the 5 fr gss . another study reported that a sheath with a smaller outer diameter was more comfortable for patients , and the threshold ratio of radial artery / sheath outer diameter was 0.9 , even in cases without radial artery occlusion and painful feelings using a conventional 5 fr sheath . for these reasons , tr access is safer and more comfortable with the 5 fr gss than conventional 5 fr sheaths . recently , the introduction of next - generation devices has made it possible to use 5 fr guiding catheters for routine pci . furthermore , the kissing balloon technique can be performed with a 5 fr guiding catheter and 0.010 in . several techniques to enhance back - up support have been introduced for complex lesions , including a child s catheter . limitations of this study include the small number of cases , single ethnic origin of the netherlands , and the use of two different hemostasis devices . especially , netherlander is well known for strong constitution of the body and the size of radial artery may be larger than that of asian race . we need more studies to other race including asian , who have small sized radial artery . because this small size of the sheath seems to be useful to people having small sized radial artery to prevent radial artery occlusion or spasm . the tr - band was mainly selected for one - day outpatient procedures because of the lack of overnight nursing staff with experience using this hemostasis device . further studies are necessary to evaluate the 5 fr gss in asian populations , which have a higher bleeding tendency . this sheath has thin wall . in this study , there were no kinking and bending phenomenon after using angiography and intervention . but we have to know that gss may be fragile with manipulating violently and have to be used gently to disturb breaking . using the echography and the doppler flow may clarify the impact of the 5fr gss on radial artery . but it was difficult to use those imaging devices , because the hospital disallowed to use it . we should make another multi center registration comparing with 5fr gss and conventional 5fr sheath in order to prove the superiority of slender sheath . the initial clinical experience with the new 5 fr gss demonstrated that it was safe and feasible for tr access . no radial artery occlusion or spasm was reported .

spatial organization , from organisms to molecules , is a defining feature of life . in multicellular organisms , cells serve to concentrate and organize biological activities , facilitating metabolism , growth , and division . in eukaryotic cells , biochemical activities can also be concentrated and regulated through the formation of non membrane - bounded , phase - separated liquid droplets such as p bodies , germ granules , nucleoli , and others ( courchaine et al . , not only is the eukaryotic nucleus segregated from the cytoplasm , but it is also spatially organized within . it has long been appreciated that chromosomes are nonrandomly arranged ( rabl , 1885 ; boveri , 1909 ) . during mitosis , chromosome condensation and alignment are critical to facilitate proper segregation ( hirano , 2015 ) . similarly , during interphase or in postmitotic cells , the spatial organization of decondensed chromosomes is tissue specific , reflecting different functional states of the genome ( parada et al . , 2004 ; meaburn and misteli , 2007 ; fraser et al . , 2015 ) . chromosomes fold and occupy characteristic regions of the interphase nucleus . in unicellular eukaryotes such as yeast , as well as in certain metazoan cells during development , chromosomes associate with the nuclear envelope , and their telomeres and centromeres cluster together at opposite sides of the nucleus ( marshall et al . , 1996 ; , 2001 ; taddei et al . , 2010 ; zimmer and fabre , 2011 ) . in differentiated cells , chromosomes tend to fold and occupy distinct , mostly nonoverlapping territories the position of chromosomes within the nucleus also correlates with cell type ; gene - rich chromosomes that are more highly transcribed tend to be positioned toward the center of the nucleus , and gene - poor chromosomes tend to be positioned toward the nuclear periphery ( parada et al . , the arrangement of chromosomes with respect to nuclear landmarks such as the nuclear periphery reflects their interaction with stable subnuclear structures . depending on the cell type , a varying but significant fraction of metazoan genomes is associated with the nuclear lamina . these lamina - associated - domains ( lads ) , up to 10 mb in length , localize at the nuclear periphery ( figure 1a ; vogel et al . , 2007 ; guelen et al . , 2008 ) . similarly , in budding yeast and fission yeast , telomeres and centromeres cluster together and associate with the nuclear envelope ( klein et al . , 1992 ; such physical interactions provide a significant constraint on chromosome folding and positioning , leading to a nonrandom arrangement of chromosomes within the nucleus . ( a ) during development , genes that are induced often move from a peripheral position in association with the lamina , where they are silenced , to a more internal site , where they are expressed ( green locus ) . ( b ) gene activation can lead to looping of genes out of their respective chromosome territories , where they can cluster together . ( c ) upon activation ( green locus ) , certain genes physically associate with the npc , causing them to localize to the nuclear periphery . thus the nuclear periphery is associated with both silencing ( red locus at the lamina ) and activation / poising ( at the npc ) . ( d ) genes that are targeted to the npc in budding yeast frequently undergo interallelic clustering and interchromosomal clustering with other genes that are targeted by the same mechanism . individual genes are often nonrandomly positioned with respect to nuclear structures and with respect to each other and their position can change upon transcriptional activation or repression . silenced loci within lads localize at the nuclear periphery , but such loci are released from the lamina and move to a more internal site upon activation ( figure 1a ; kosak et al . , 2002 ; zink et al . , 2004 ; meister et al . , 2010 ; similarly , transcriptional induction can lead to a locus looping out from its chromosome territory ( figure 1b ; noordermeer et al . , heat shock genes reposition near stress granules upon induction ( khanna et al . , 2014 ) . furthermore , in budding yeast , drosophila , caenorhabditis elegans , and mammalian cells , many active genes physically interact with nuclear pore proteins ( casolari et al . , 2004 , 2005 ; , 2008 ; capelson et al . , 2010 ; kalverda and fornerod , 2010 ; liang et al . , 2013 ; rohner et al . , these interactions apparently occur exclusively at the nuclear periphery ( with the nuclear pore complex [ npc ] ; figure 1c ) , in flies and mammals , these interactions can occur either at the nuclear periphery or in the nucleoplasm ( with soluble nuclear pore proteins ; capelson et al . interaction with the npc leads to positioning at the nuclear periphery ( figure 1c ) . thus changes in gene expression are frequently coupled to movement of loci with respect to nuclear landmarks . coregulated genes sometimes cluster together through interchromosomal interactions ( figure 1 , b and d ) . clustering has been observed between genes induced during erythropoiesis ( brown et al . , 2006 ; schoenfelder et al . , 2010 ) , genes induced by estrogen ( lin et al . , 2009 ) , and genes induced by infection ( apostolou and thanos , 2008 ) . in yeasts , both trna genes and genes that are targeted to the npc undergo interchromosomal clustering ( figure 1d ; thompson et al . , 2003 ; noma et al . , 2006 ; brickner et al . , these observations suggest that the nucleus might be functionally segregated by creating clusters of genes of related function or with related modes of regulation . nonrandom spatial organization in some cases represents an emergent phenomenon , a functional outcome of transcription , chromatin structure , or polymer biophysics ( misteli , 2008 , 2009 ) . emergent phenomena resulting from self - organization are common and extremely important in biology , and this is a critical consideration in studying nuclear cell biology . interpreting genetic perturbation of emergent phenomena is more difficult than interpreting genetic perturbation of active targeting mechanisms . for example , if the biophysical process of transcription , chromatin decompaction , or mrna processing were more energetically costly in the more densely compacted environment within a chromosome territory , this might result in the movement of active genes to the edge of the territory . this movement , however , would not represent active , specific targeting to the interterritorial space . indeed , modeling chromosomes as polymers and incorporating simple biophysical differences between euchromatin and heterochromatin can recapitulate formation of chromosome territories and the localization of active genes between chromosome territories ( cook and marenduzzo , 2009 ; heermann , 2011 ; wang et al . , 2015 ) . similarly , some subnuclear structures are not stable landmarks but are built around the genes with which they colocalize . for example , nucleoli and histone locus bodies colocalize with the ribosomal dna and histone genes , respectively . however , such bodies can be initiated to form at an ectopic locus by tethering nucleating factors to that site ( karpen et al . , this type of spatial organization , although specific to particular genes and giving rise to a highly biased pattern , reflects an emergent property of certain genes rather than active positioning of genes near nuclear bodies . distinguishing between nonrandom arrangements that are produced by active targeting mechanisms and i focus on cases in which the nonrandom positioning of genes and chromosomal domains appears to be conferred by active targeting mechanisms . to define active mechanisms , active mechanisms that control subnuclear positioning ought to exhibit specificity and depend on information that is necessary and/or sufficient to promote targeting to stable sites . nonrandom spatial organization in some cases represents an emergent phenomenon , a functional outcome of transcription , chromatin structure , or polymer biophysics ( misteli , 2008 , 2009 ) . emergent phenomena resulting from self - organization are common and extremely important in biology , and this is a critical consideration in studying nuclear cell biology . interpreting genetic perturbation of emergent phenomena is more difficult than interpreting genetic perturbation of active targeting mechanisms . for example , if the biophysical process of transcription , chromatin decompaction , or mrna processing were more energetically costly in the more densely compacted environment within a chromosome territory , this might result in the movement of active genes to the edge of the territory . this movement , however , would not represent active , specific targeting to the interterritorial space . indeed , modeling chromosomes as polymers and incorporating simple biophysical differences between euchromatin and heterochromatin can recapitulate formation of chromosome territories and the localization of active genes between chromosome territories ( cook and marenduzzo , 2009 ; heermann , 2011 ; wang et al . , 2015 ) . similarly , some subnuclear structures are not stable landmarks but are built around the genes with which they colocalize . for example , nucleoli and histone locus bodies colocalize with the ribosomal dna and histone genes , respectively . however , such bodies can be initiated to form at an ectopic locus by tethering nucleating factors to that site ( karpen et al . , 1988 ; kaiser et al . , 2008 ; mao et al . , 2011 ; shevtsov and dundr , 2011 ) . this type of spatial organization , although specific to particular genes and giving rise to a highly biased pattern , reflects an emergent property of certain genes rather than active positioning of genes near nuclear bodies . distinguishing between nonrandom arrangements that are produced by active targeting mechanisms and those that are produced by emergent phenomena is critical to understanding nuclear architecture . here i focus on cases in which the nonrandom positioning of genes and chromosomal domains appears to be conferred by active targeting mechanisms . to define active mechanisms , active mechanisms that control subnuclear positioning ought to exhibit specificity and depend on information that is necessary and/or sufficient to promote targeting to stable sites . if spatial organization of the genome is controlled by active mechanisms , then what is the source of the information ? is spatial organization encoded by the dna sequence , chromatin changes ( potentially independent of the dna sequence ) , or a combination of both ? work from several experimental systems reveals that both sequence - specific dna - binding proteins and chromatin modifications play essential roles in controlling the spatial organization of genes and chromosomal domains . binding sites for sequence - specific dna - binding proteins such as ctcf and transcription factors serve as the genetic information that affects chromosome folding and spatial positioning . non genetic chromatin changes such as histone methylation and variant histones also play essential roles in controlling the positioning of individual genes and chromosomal domains within the nucleus . spatial positioning of individual genes with respect to stable nuclear landmarks can be controlled by cis - acting dna elements that recruit sequence - specific dna - binding proteins . in drosophila , the gypsy insulator element is sufficient to induce targeting of a locus to the nuclear periphery ( gerasimova et al . , 2000 ) . for developmentally induced genes that localize at the nuclear periphery in association with the nuclear lamina before induction ( kosak et al . , 2002 ) , the positioning at the periphery can be determined by cis - acting dna sequences . for example , insertion of the igh and cyp3a genes at ectopic sites in the genome is sufficient to reposition the ectopic locus to the nuclear periphery ( zullo et al . , 2012 ) . the transcription factors ckrox and yy1 are critical for this targeting ( zullo et al . , 2012 ; harr et al . , 2015 ) . similarly , the -globin gene possesses cis - acting dna elements that are necessary for targeting to the nuclear periphery ( bian et al . , 2013 ) . binding sites play a critical role in targeting to the nuclear lamina ( figure 2a ) . ( a ) targeting of genes to the nuclear lamina requires both transcription factors such as c - krox and yy1 and methylation of histone h3 on lysine 9 ( red circles ) . in c. elegans ( b ) targeting to the npc ( or interaction with nuclear pore proteins in the nucleoplasm of drosophila or mammals ) requires transcription factors and , in some cases , chromatin changes . several yeast transcription factors are both necessary and sufficient to cause targeting to the npc . however , histone acetylation , h3k4 methylation ( green circles ) , and h2a.z incorporation ( green nucleosomes ) are also required in certain cases . hundreds of genes that interact with nuclear pore proteins have been identified in yeast , flies , and mammalian cells ( casolari et al . initially , it was believed that this may represent an emergent product of transcription by which npc - associated mrna export factors might create an affinity sink for actively transcribed genes ( casolari et al . , 2005 ; dieppois et al . , 2006 ; taddei et al . , however , targeting to the npc in yeast does not require active transcription ( schmid et al . , 2007 , 2016 ; ahmed et al . , 2010 ; light et al . , 2010 ; randise - hinchliff et al . , , binding sites for certain transcription factors are enriched among genes that interact with pore proteins ( casolari et al . , 2008 ; liang et al . , 2013 ) . in drosophila , gaga factor binding sites are also enriched for nuclear pore protein binding , and the transcription factor mbd - r2 physically interacts with the nuclear pore protein nup98 and promotes interaction of nup98 with target genes ( capelson et al . , 2010 ; kalverda et al . , 2010 ; pascual - garcia et al . , however , in most cases , the functional significance of these transcription factors in controlling interaction with nuclear pore proteins or spatial positioning of target genes has not yet been tested . in budding yeast , binding sites have been functionally identified as both necessary and sufficient to target genes to the nuclear periphery ( ahmed et al . , 2010 ; light et al . , 2010 ; brickner et al . , 2012 , 2016 ; durso et al . , 2016 ; randise - hinchliff et al . , not every yeast transcription factor has this ability , suggesting that a subset of transcription factors affects gene positioning . five transcription factors have been identified ( put3 , cbf1 , gcn4 , ste12 , and sfl1 ) that are both necessary for targeting endogenous loci to the periphery and whose binding sites function as dna zip codes that are sufficient to target an ectopic site to the periphery ( figure 2b ) . in several of these cases ( gcn4 , ste12 , and sfl1 ) , the transcription factors cause peripheral targeting when tethered to an ectopic site through a heterologous dna - binding domain , confirming that they are indeed sufficient to confer positioning at the periphery ( durso et al . , 2016 ; this suggests that controlling gene positioning is an underappreciated function of some transcription factors ( figure 2b ) . transcription factors and their binding sites also control interchromosomal clustering of coregulated genes . before differentiation of t helper cells , the interferon gene clusters with a locus control region ( lcr ) on another chromosome , and this is regulated by the dnase - hypersensitive region of the lcr ( spilianakis et al . , 2005 ) . clustering of the interferon gene with sites on other chromosomes in response to virus infection requires the nf-b transcription factor ( apostolou and thanos , 2008 ) . clustering of coregulated genes during erythropoiesis requires the klf1 transcription factor ( schoenfelder et al . , 2010 ) , and clustering of bacterial artificial chromosomes carrying the hsp70 gene near nuclear speckles requires the hsp70 promoter ( hu et al . , 2010 ) . similarly , clustering of trna genes in both budding yeast and fission yeast requires the sequence - specific transcription factor tfiiic ( noma et al . , 2006 ; transcription factors that control positioning to the nuclear periphery are also necessary for specific interchromosomal clustering in association with the nuclear pore ( brickner et al . , 2012 , 2015 ; 2016 ) . of importance , insertion of their dna - binding sites at an ectopic site in the genome can promote clustering with endogenous genes that possess the same binding site ( brickner et al . this suggests that genomes encode both peripheral localization and interchromosomal clustering through cis - acting dna elements such as transcription factor binding sites . chromatin modifications often correlate with spatial position and , in some cases , are required for the normal spatial arrangement of the genome . histone deacetylation recruits sir4 , which binds to both hypoacetylated histones and the nuclear envelope membrane protein esc1 , providing a physical link between chromatin state and a subnuclear structure ( taddei and gasser , 2012 ) . lamina - associated domains tend to be transcriptionally repressed and methylated on histone h3 lysine 9 ( guelen et al . , 2008 ; wen et al . , 2009 ; yokochi et al . , 2009 ; kind et al . , 2013 ) loss of the h3k9 methyl transferase g9a leads to loss of peripheral h3k9me2 but does not obviously alter the peripheral localization of all genes ( yokochi et al . , however , association of lamina - targeted bacs or endogenous lads in human cells or lamina - associated repetitive arrays in c. elegans is dependent on h3k9 methylation ( towbin et al . thus , together with the transcription factors mentioned earlier , h3k9 methylation is required for the interaction with certain loci with the nuclear periphery ( figure 2a ) . in c. elegans , the system in which this requirement was discovered used long arrays of transgenes ( meister et al . , 2010 ) . the h3k9 methylation of these arrays is apparently a nonspecific product of their repetitive nature . targeting to the nuclear lamina and h3k9 methylation are mutually reinforcing events : h3k9 methylation is both promoted by targeting to the nuclear lamina and required for targeting to the lamina . a nuclear envelope associated protein called cec-4 binds to methylated h3k9 through a chromodomain to mediate localization of heterochromatic repetitive arrays to the nuclear lamina ( gonzalez - sandoval et al . this suggests that h3k9 methylation alone can target such arrays to the nuclear lamina , independent of their sequence and presumably independent of dna - binding proteins . this requires the polycomb group protein pc ( lanzuolo et al . , 2007 ) . however , mutations that disrupt polycomb response elements , which reduce recruitment of polycomb group proteins , do not strongly affect either h3k27me3 or polycomb body formation . this suggests that h3k27 methylation , rather than a dna - binding protein , may facilitate the formation of polycomb bodies . targeting of certain yeast genes to the npc requires the saga histone acetyltransferase and is inhibited by the rpd3(l ) histone deacetylase ( cabal et al . , 2006 ; luthra et al . , 2007 ; furthermore , targeting of yeast ino1 to the nuclear periphery after repression ( a phenomenon called epigenetic transcriptional memory ) requires both local incorporation of h2a.z and dimethylation of h3k4 ( brickner et al . therefore chromatin modifications also play essential roles in targeting to the nuclear pore complex ( figure 2b ) . in addition to active and poised genes , the npc in budding yeast and drosophila also interacts with persistent double - strand breaks , eroded telomeres , and collapsed replication forks , and this interaction facilitates dna repair ( nagai et al . , 2008 ; kalocsay et al . , 2009 ; ryu et al . , relocalization of dsbs to the npc requires both h2a.z and sumoylation of either chromatin or repair factors , leading to a physical interaction with the sumo - dependent ubiquitin ligase slx5/slx8 , which interacts with the npc component nup84 ( nagai et al . , 2008 ; thus positioning of dna damage , which is unlikely to be dependent on dna sequence , is mediated by changes in chromatin and associated proteins . because transcription factors and chromatin changes influence each other and can have indirect effects , in most cases , it remains to be determined whether either of these mechanisms alone is sufficient to confer spatial positioning . furthermore , the function of transcription factors themselves might be regulated by posttranslational modifications such as acetylation and sumoylation ( texari et al . , 2013 ) . in cases in which both dna - binding proteins and chromatin modifications play essential roles in controlling the spatial organization of the genome ( i.e. , neither is sufficient ) , they might function in the same linear pathway or by a combinatorial mechanism . the requirement for histone acetylation or methylation in targeting genes to the npc ( figure 2b ) may represent either a direct role for chromatin in mediating targeting to the npc or a role for chromatin in regulating transcription factor binding / function . in support of this idea , rpd3(l ) regulates put3 dna binding , and saga is required for targeting of certain genes to the periphery because histone acetylation promotes binding of transcription factors . in support of this idea , saga is required for peripheral targeting of genes whose subnuclear localization is regulated by transcription factor occupancy but is not required for peripheral targeting of genes whose subnuclear localization is regulated downstream of transcription factor occupancy ( randise - hinchliff et al . , this suggests that histone acetylation ( or other functions of saga ) may regulate transcription factor occupancy by regulating either dna binding or transcription factor abundance . the histone modifications associated with transcriptional memory are necessary but not sufficient to confer targeting to the nuclear periphery . mutants lacking h2a.z or unable to methylate h3k4 fail to target genes to the npc during memory ( brickner et al . however , neither h2a.z nor h3k4me2 is sufficient to cause targeting to the npc ( light et al . , 2010 ; this suggests that targeting to the npc requires both a transcription factor and the correct chromatin state and that they may function together ( figure 2b ) . future work will distinguish a direct role for chromatin in affecting gene positioning in this system from a regulatory role . spatial positioning of individual genes with respect to stable nuclear landmarks can be controlled by cis - acting dna elements that recruit sequence - specific dna - binding proteins . in drosophila , the gypsy insulator element is sufficient to induce targeting of a locus to the nuclear periphery ( gerasimova et al . , 2000 ) . for developmentally induced genes that localize at the nuclear periphery in association with the nuclear lamina before induction ( kosak et al . , 2002 ) , the positioning at the periphery can be determined by cis - acting dna sequences . for example , insertion of the igh and cyp3a genes at ectopic sites in the genome is sufficient to reposition the ectopic locus to the nuclear periphery ( zullo et al . , the transcription factors ckrox and yy1 are critical for this targeting ( zullo et al . , 2012 ; harr et al . , similarly , the -globin gene possesses cis - acting dna elements that are necessary for targeting to the nuclear periphery ( bian et al . , 2013 ) . binding sites play a critical role in targeting to the nuclear lamina ( figure 2a ) . ( a ) targeting of genes to the nuclear lamina requires both transcription factors such as c - krox and yy1 and methylation of histone h3 on lysine 9 ( red circles ) . in c. elegans ( b ) targeting to the npc ( or interaction with nuclear pore proteins in the nucleoplasm of drosophila or mammals ) requires transcription factors and , in some cases , chromatin changes . several yeast transcription factors are both necessary and sufficient to cause targeting to the npc . however , histone acetylation , h3k4 methylation ( green circles ) , and h2a.z incorporation ( green nucleosomes ) are also required in certain cases . hundreds of genes that interact with nuclear pore proteins have been identified in yeast , flies , and mammalian cells ( casolari et al . initially , it was believed that this may represent an emergent product of transcription by which npc - associated mrna export factors might create an affinity sink for actively transcribed genes ( casolari et al . however , targeting to the npc in yeast does not require active transcription ( schmid et al . , 2006 ; brickner et al . , 2007 , 2016 ; ahmed et al . , 2010 ; light et al . , 2010 ; randise - hinchliff et al . , , binding sites for certain transcription factors are enriched among genes that interact with pore proteins ( casolari et al . , 2004 , 2005 ; , 2008 ; liang et al . , 2013 ) . in drosophila , gaga factor binding sites are also enriched for nuclear pore protein binding , and the transcription factor mbd - r2 physically interacts with the nuclear pore protein nup98 and promotes interaction of nup98 with target genes ( capelson et al . , 2010 ; kalverda et al . , 2010 ; however , in most cases , the functional significance of these transcription factors in controlling interaction with nuclear pore proteins or spatial positioning of target genes has not yet been tested . in budding yeast , binding sites have been functionally identified as both necessary and sufficient to target genes to the nuclear periphery ( ahmed et al . , 2010 ; light et al . , 2010 ; brickner et al . , 2012 , 2016 ; durso et al . , 2016 ; not every yeast transcription factor has this ability , suggesting that a subset of transcription factors affects gene positioning . five transcription factors have been identified ( put3 , cbf1 , gcn4 , ste12 , and sfl1 ) that are both necessary for targeting endogenous loci to the periphery and whose binding sites function as dna zip codes that are sufficient to target an ectopic site to the periphery ( figure 2b ) . in several of these cases ( gcn4 , ste12 , and sfl1 ) , the transcription factors cause peripheral targeting when tethered to an ectopic site through a heterologous dna - binding domain , confirming that they are indeed sufficient to confer positioning at the periphery ( durso et al . , 2016 ; randise - hinchliff et al . , this suggests that controlling gene positioning is an underappreciated function of some transcription factors ( figure 2b ) . transcription factors and their binding sites also control interchromosomal clustering of coregulated genes . before differentiation of t helper cells , the interferon gene clusters with a locus control region ( lcr ) on another chromosome , and this is regulated by the dnase - hypersensitive region of the lcr ( spilianakis et al . , 2005 ) . clustering of the interferon gene with sites on other chromosomes in response to virus infection requires the nf-b transcription factor ( apostolou and thanos , 2008 ) . clustering of coregulated genes during erythropoiesis requires the klf1 transcription factor ( schoenfelder et al . , 2010 ) , and clustering of bacterial artificial chromosomes carrying the hsp70 gene near nuclear speckles requires the hsp70 promoter ( hu et al . , 2010 ) . similarly , clustering of trna genes in both budding yeast and fission yeast requires the sequence - specific transcription factor tfiiic ( noma et al . transcription factors that control positioning to the nuclear periphery are also necessary for specific interchromosomal clustering in association with the nuclear pore ( brickner et al . , 2012 , 2015 ; 2016 ) . of importance , insertion of their dna - binding sites at an ectopic site in the genome can promote clustering with endogenous genes that possess the same binding site ( brickner et al . this suggests that genomes encode both peripheral localization and interchromosomal clustering through cis - acting dna elements such as transcription factor binding sites . chromatin modifications often correlate with spatial position and , in some cases , are required for the normal spatial arrangement of the genome . histone deacetylation recruits sir4 , which binds to both hypoacetylated histones and the nuclear envelope membrane protein esc1 , providing a physical link between chromatin state and a subnuclear structure ( taddei and gasser , 2012 ) . lamina - associated domains tend to be transcriptionally repressed and methylated on histone h3 lysine 9 ( guelen et al . , 2008 ; wen et al . , 2009 ; yokochi et al . , 2009 ; kind et al . , 2013 ) . loss of the h3k9 methyl transferase g9a leads to loss of peripheral h3k9me2 but does not obviously alter the peripheral localization of all genes ( yokochi et al . , however , association of lamina - targeted bacs or endogenous lads in human cells or lamina - associated repetitive arrays in c. elegans is dependent on h3k9 methylation ( towbin et al . thus , together with the transcription factors mentioned earlier , h3k9 methylation is required for the interaction with certain loci with the nuclear periphery ( figure 2a ) . in c. elegans , the system in which this requirement was discovered used long arrays of transgenes ( meister et al . , 2010 ) . the h3k9 methylation of these arrays is apparently a nonspecific product of their repetitive nature . targeting to the nuclear lamina and h3k9 methylation are mutually reinforcing events : h3k9 methylation is both promoted by targeting to the nuclear lamina and required for targeting to the lamina . a nuclear envelope associated protein called cec-4 binds to methylated h3k9 through a chromodomain to mediate localization of heterochromatic repetitive arrays to the nuclear lamina ( gonzalez - sandoval et al . , 2015 ) . this suggests that h3k9 methylation alone can target such arrays to the nuclear lamina , independent of their sequence and presumably independent of dna - binding proteins . this requires the polycomb group protein pc ( lanzuolo et al . , 2007 ) . however , mutations that disrupt polycomb response elements , which reduce recruitment of polycomb group proteins , do not strongly affect either h3k27me3 or polycomb body formation . this suggests that h3k27 methylation , rather than a dna - binding protein , may facilitate the formation of polycomb bodies . targeting of certain yeast genes to the npc requires the saga histone acetyltransferase and is inhibited by the rpd3(l ) histone deacetylase ( cabal et al . , 2006 ; luthra et al . , 2007 ; furthermore , targeting of yeast ino1 to the nuclear periphery after repression ( a phenomenon called epigenetic transcriptional memory ) requires both local incorporation of h2a.z and dimethylation of h3k4 ( brickner et al . , 2007 ; light et al . , 2010 , 2013 ) . therefore chromatin modifications also play essential roles in targeting to the nuclear pore complex ( figure 2b ) . in addition to active and poised genes , the npc in budding yeast and drosophila also interacts with persistent double - strand breaks , eroded telomeres , and collapsed replication forks , and this interaction facilitates dna repair ( nagai et al . , 2008 ; relocalization of dsbs to the npc requires both h2a.z and sumoylation of either chromatin or repair factors , leading to a physical interaction with the sumo - dependent ubiquitin ligase slx5/slx8 , which interacts with the npc component nup84 ( nagai et al . , 2008 ; kalocsay et al . , 2009 ; churikov et al . , 2016 ; horigome et al . , thus positioning of dna damage , which is unlikely to be dependent on dna sequence , is mediated by changes in chromatin and associated proteins . because transcription factors and chromatin changes influence each other and can have indirect effects , in most cases , it remains to be determined whether either of these mechanisms alone is sufficient to confer spatial positioning . furthermore , the function of transcription factors themselves might be regulated by posttranslational modifications such as acetylation and sumoylation ( texari et al . , 2013 ) . in cases in which both dna - binding proteins and chromatin modifications play essential roles in controlling the spatial organization of the genome ( i.e. , neither is sufficient ) , they might function in the same linear pathway or by a combinatorial mechanism . the requirement for histone acetylation or methylation in targeting genes to the npc ( figure 2b ) may represent either a direct role for chromatin in mediating targeting to the npc or a role for chromatin in regulating transcription factor binding / function . in support of this idea , rpd3(l ) regulates put3 dna binding , and saga is required for targeting of certain genes to the periphery because histone acetylation promotes binding of transcription factors . in support of this idea , saga is required for peripheral targeting of genes whose subnuclear localization is regulated by transcription factor occupancy but is not required for peripheral targeting of genes whose subnuclear localization is regulated downstream of transcription factor occupancy ( randise - hinchliff et al . , 2016 ) . this suggests that histone acetylation ( or other functions of saga ) may regulate transcription factor occupancy by regulating either dna binding or transcription factor abundance . the histone modifications associated with transcriptional memory are necessary but not sufficient to confer targeting to the nuclear periphery . mutants lacking h2a.z or unable to methylate h3k4 fail to target genes to the npc during memory ( brickner et al . however , neither h2a.z nor h3k4me2 is sufficient to cause targeting to the npc ( light et al . , 2010 ; durso et al . , 2016 this suggests that targeting to the npc requires both a transcription factor and the correct chromatin state and that they may function together ( figure 2b ) . future work will distinguish a direct role for chromatin in affecting gene positioning in this system from a regulatory role . future work will seek to develop a more complete understanding of the molecular and evolutionary mechanisms that control the spatial organization of the genome . to make progress , first , we must distinguish spatial organization that is a product of emergent properties of nuclear biology from spatial organization that is the product of active mechanisms . both types of phenomena contribute to the spatial organization of the genome , and they need to be integrated to understand the whole . second , we must carefully define the relationship between dna - binding proteins and chromatin changes in determining spatial organization . subnuclear positioning of genes and domains is controlled by dna - binding proteins , chromatin structure , and , in some cases , both . understanding the evolutionary processes that can select for such mechanisms poses an exciting and important problem for future work .

binocular surgery is the most commonly used approach for surgical treatment of large - angle horizontal strabismus involving three or four horizontal rectus muscles . this procedure is widely used because it avoids significant limitations of ocular movement , which could occur in surgeries of greater magnitude.15 however , monocular surgery has many advantages , such as preserving some muscles if a repeat operation is required , avoiding the exposure of the dominant eye to the inherent risks of a surgical procedure and reducing surgical time.610 furthermore , a monocular procedure can be performed in adults under peribulbar anesthesia , which is associated with decreased morbidity and mortality when compared with general anesthesia , and also offers swifter recovery and lower incidence rates of nausea and vomiting.1119 the aim of this study was to evaluate the results of monocular surgery under peribulbar anesthesia for large - angle horizontal strabismus . we evaluated the medical records of 92 consecutive patients who underwent monocular surgery under peribulbar anesthesia after january 2004 at the department of ophthalmology , university of campinas ( campinas , sp , brazil ) . the inclusion criteria were : - large - angle strabismus : defined , in this study , as an angle of deviation of 40 prism diopters ( pd ) or greater;- primary strabismus , excluding all cases of paretic , restrictive or consecutive strabismus . patients who had previously been administered botulinum toxin a to treat strabismus were also excluded.- horizontal strabismus ( esotropia or exotropia ) , excluding any cases with associated vertical deviation.- follow - up data lasting at least 6 months . - large - angle strabismus : defined , in this study , as an angle of deviation of 40 prism diopters ( pd ) or greater ; - primary strabismus , excluding all cases of paretic , restrictive or consecutive strabismus . patients who had previously been administered botulinum toxin a to treat strabismus were also excluded . - horizontal strabismus ( esotropia or exotropia ) , excluding any cases with associated vertical deviation . - follow - up data lasting at least 6 months . complete ophthalmologic examination ( best - corrected visual acuity measurement , biomicroscopy , tonometry and indirect ophthalmoscopy ) . measurement of angle of deviation the angle of strabismus was measured in prism diopters ( pd ) , with the best optic correction , in all positions , for both distance and near vision . for patients with good bilateral visual acuity , this parameter was measured by the prism and cover test using a suitable fixation target . patients with poor vision or profound amblyopia were submitted to krinsky measurement , based on the corneal reflex . 2022 after evaluation of strabismus , the subjects were divided into group 1 ( patients with esotropia ) and group 2 ( patients with exotropia ) . surgical planning and surgery the surgeries consisted of recession and resection and are described in table 1 ( group 1 ) and table 2 ( group 2 ) . peribulbar anesthesia was performed by the ocular surgeon , and all patients received 5 mg of diazepam orally , thirty minutes before anesthesia . an intravenous line , oxygen nasal cannula , cardiac monitoring and continuous pulse oximetry were employed . the local anesthetic consisted of 0.5% bupivacaine without epinephrine and 2% lidocaine with epinephrine . patients were examined at least on the first , seventh and thirtieth days and three and six months after surgery . measurements of angles of deviation were recorded during all visits , but the data considered in this study were from the sixth postoperative month only . second surgical procedure in cases of residual deviations of over 15 pd in the sixth month , a second procedure in the dominant eye was indicated , and the follow - up continued until at least six months after the second surgery . all patients accepted the conditions when informed about the surgery and possible need for a second surgery . the data were analyzed using the mann - whitney test for comparison of age and preoperative and postoperative angles of deviation between the groups ; repeated - measures anova ( analysis of variance ) for preoperative and postoperative comparisons ; and ancova s ( analysis of covariance ) to adjust for covariates of age and type of deviation ( esotropia or exotropia ) . the roc curve ( receiver operating characteristic ) was used to define the cutoff point of preoperative angle of deviation for a successful outcome the statistical analysis was performed using the sas system for windows ( statistical analysis system ) , version 8.01 , 19992000 . this study was approved by the research ethics committee of the faculty of medical sciences - university of campinas , campinas / sp , brazil . we evaluated the medical records of 92 consecutive patients who underwent monocular surgery under peribulbar anesthesia after january 2004 at the department of ophthalmology , university of campinas ( campinas , sp , brazil ) . the inclusion criteria were : - large - angle strabismus : defined , in this study , as an angle of deviation of 40 prism diopters ( pd ) or greater;- primary strabismus , excluding all cases of paretic , restrictive or consecutive strabismus . patients who had previously been administered botulinum toxin a to treat strabismus were also excluded.- horizontal strabismus ( esotropia or exotropia ) , excluding any cases with associated vertical deviation.- follow - up data lasting at least 6 months . - large - angle strabismus : defined , in this study , as an angle of deviation of 40 prism diopters ( pd ) or greater ; - primary strabismus , excluding all cases of paretic , restrictive or consecutive strabismus . patients who had previously been administered botulinum toxin a to treat strabismus were also excluded . - horizontal strabismus ( esotropia or exotropia ) , excluding any cases with associated vertical deviation . - follow - up data lasting at least 6 months . complete ophthalmologic examination ( best - corrected visual acuity measurement , biomicroscopy , tonometry and indirect ophthalmoscopy ) . measurement of angle of deviation the angle of strabismus was measured in prism diopters ( pd ) , with the best optic correction , in all positions , for both distance and near vision . for patients with good bilateral visual acuity , this parameter was measured by the prism and cover test using a suitable fixation target . patients with poor vision or profound amblyopia were submitted to krinsky measurement , based on the corneal reflex . 2022 after evaluation of strabismus , the subjects were divided into group 1 ( patients with esotropia ) and group 2 ( patients with exotropia ) . surgical planning and surgery the surgeries consisted of recession and resection and are described in table 1 ( group 1 ) and table 2 ( group 2 ) . peribulbar anesthesia was performed by the ocular surgeon , and all patients received 5 mg of diazepam orally , thirty minutes before anesthesia . an intravenous line , oxygen nasal cannula , cardiac monitoring and continuous pulse oximetry were employed . the local anesthetic consisted of 0.5% bupivacaine without epinephrine and 2% lidocaine with epinephrine . patients were examined at least on the first , seventh and thirtieth days and three and six months after surgery . measurements of angles of deviation were recorded during all visits , but the data considered in this study were from the sixth postoperative month only . second surgical procedure in cases of residual deviations of over 15 pd in the sixth month , a second procedure in the dominant eye was indicated , and the follow - up continued until at least six months after the second surgery . all patients accepted the conditions when informed about the surgery and possible need for a second surgery . the data were analyzed using the mann - whitney test for comparison of age and preoperative and postoperative angles of deviation between the groups ; repeated - measures anova ( analysis of variance ) for preoperative and postoperative comparisons ; and ancova s ( analysis of covariance ) to adjust for covariates of age and type of deviation ( esotropia or exotropia ) . the roc curve ( receiver operating characteristic ) was used to define the cutoff point of preoperative angle of deviation for a successful outcome . the statistical analysis was performed using the sas system for windows ( statistical analysis system ) , version 8.01 , 19992000 . this study was approved by the research ethics committee of the faculty of medical sciences - university of campinas , campinas / sp , brazil . forty - four patients had esotropia ( group 1 ) and forty - eight had exotropia ( group 2 ) . the patients ages , as well as other data , such as preoperative angles of deviation , surgical procedures and postoperative deviations are shown in tables 1 ( patients with esotropia ) and 2 ( patients with exotropia ) . all patients with preoperative deviation of up to 60 pd in both groups underwent successful surgeries ( postoperative deviation of 15 pd or less in the sixth postoperative month ) . in patients with 65 pd preoperative deviations , one of three patients in group 1 and five of nine patients in group 2 underwent successful surgeries . all patients with deviations over 65 pd presented with a residual deviation of over 15 pd , which ranged from 20 to 55 pd in group 1 , and from 20 to 30 pd in group 2 . a total of 25 patients exhibited residual deviations of over 15 pd , but only thirteen ( 52% ) elected to undergo a second procedure , which turned out to be successful in all cases . the angle of deviation after the first surgical procedure , the second surgical procedure and the postoperative deviation six months after surgery are shown in tables 3 ( patients with esotropia ) and 4 ( patients with exotropia ) . no patient presented with severe limitations in respect of ocular movement . however , all patients who underwent the 8 mm recession of medial rectus in group 1 experienced some degree of limitation in ocular movement . in group 2 , 6 of the 10 patients submitted to 9 mm recession of lateral rectus presented with some degree of limited ocular movement . based on the mann - whitney test , there was a statistically significant difference in age ( group 1 was younger , p=0.026 ) , but no difference in the preoperative angle of deviation ( p=0.0512 ) , the magnitude of surgical correction ( p=0.6602 ) and the postoperative results ( p=0.320 ) . the preoperative deviations were significantly greater than the postoperative in both groups ( anova , p<0.0001 ) . with ancova , our analyses were adjusted for the variables of age and type of strabismus ( esotropia or exotropia ) , and there were no statistically significant differences ( p= 0.217 ) . the spearman s rank correlation coefficient showed no correlation between age and postoperative deviation ( r = 0.13360 , p = 0.3047 ) . there was a moderate correlation between preoperative and postoperative deviations ( r = 0.43703 , p = 0.0004 ) . the roc analysis ( figure 1 ) showed that the cutoff point of the preoperative angle for obtaining a successful surgery result was 62.5 pd . the sensitivity and specificity using this cutoff point , the estimated area under roc curve and the 95% confidence interval are shown in figure 1 . most studies of surgical procedures in large - angle horizontal strabismus were conducted in congenital esotropia . several authors reported high rates of success , from 70 to 91% , in large - angle congenital esotropia with large medial rectus recessions , and no significant adduction limitations.2331 more recently , vroman et al . and prieto - diaz and souza - dias also suggested that bilateral medial rectus recession was the preferred treatment for large - angle congenital esotropia.30,31 other studies , however , suggest that interventions involving three or four muscles are more effective and avoid convergence injuries.35 several studies of large - angle exotropia have reported success rates ranging from 72% to 80% in bilateral lateral rectus recessions , without significant abduction limitations.610 berland et al . reported a success rate of 80% with a 8 to 9 mm bilateral lateral rectus recession , but with abduction limitations in 30% of the 24 patients studied.6 bracamontes et al . supported bilateral surgery in large - angle exotropias in patients with low vision in one or both eyes , because the relapse rate is higher with monocular surgery.32 however , currie et al . suggested that interventions involving three or four muscles in large - angle exotropias are more effective than surgery involving only two muscles.2 the roc analysis was used to define the cutoff point of the preoperative deviation , in order to achieve a successful result ( 62.5 pd ) corresponding to the area under the curve with a 95% confidence interval . we identified no differences between groups 1 and 2 because the ancova test did not show any influence of the type of strabismus on the postoperative result . there have been no other studies to date that have estimated a cutoff point in terms of the preoperative deviation needed to achieve a successful outcome in monocular surgery for large - angle strabismus . our results show that , after the second procedure , the residual deviations achieved results of 10 pd or less , suggesting that strabismus with a preoperative angle of deviation of 65 pd or greater ( tables 3 and 4 ) exhibited the best outcomes following binocular approaches . ocular movement may be limited in major recessions and in patients with good bilateral visual acuity ; these limitations may cause damage in terms of the convergence amplitude . most authors recommend not exceeding 7 mm medial rectus recession and 8 mm lateral rectus recession to avoid ocular movement limitations . in this study , only 8 mm medial rectus recession in group 1 and 9 mm lateral rectus recession in group 2 were associated with some degree of limitation . these results confirm the conclusions from other studies , all of which recommend avoiding large recessions.29 , 31 the effects of peribulbar anesthesia in terms of surgical results have been previously reported by our group . our published studies compared the postoperative results of strabismus surgery performed under peribulbar and general anesthesia and confirmed that the type of anesthesia used did not impact the surgical results.33,34 the disadvantages of this type of anesthesia include reduced globe mobility , which causes problems in analyzing passive stiffness . ocular complications of peribulbar anesthesia include globe perforation , retrobulbar hemorrhage , ptosis , optic nerve injury and central retinal artery occlusion35,36 however , these complications are rare and occurred only in 0.006% of 16,224 consecutive peribulbar block cases in a multicenter study.36 monocular surgery under peribulbar anesthesia can be a viable alternative for large - angle horizontal strabismus for patients who present deviations of up to 60 pd .

the term subclinical hypothyroidism is used to define that grade of primary hypothyroidism , in which there is an elevated thyroid - stimulating hormone ( tsh ) concentration in the presence of normal serum free thyroxine ( t4 ) and triiodothyronine concentrations . the worldwide prevalence of spontaneous overt hypothyroidism is between 1% and 2% and ten times more common in women than in men while approximately 8% of women and 3% men have subclinical hypothyroidism . the prevalence of overt hypothyroidism has been reported between 3.5% and 4.2% while subclinical hypothyroidism has been reported in 8.02%19.3% of population in various studies across india . globally , thyroid disorders continue to be common yet one of the most under - diagnosed and neglected chronic health conditions . patient 's knowledge and awareness about the disease and its treatment is very important for good long - term outcome and compliance in any chronic disease . studies have shown the importance of improving patient 's knowledge through education and associated benefits of improving compliance with health - care appointments and medications for patients with hypertension and diabetes although such data are scarce in the context of hypothyroidism . such studies on hypothyroidism can help the physician to concentrate on specific issues during their first interaction with the patients as well as during follow - up . there is a paucity of data on the knowledge , awareness , and practices ( kap ) among the primary hypothyroidism patients in the indian population . the present study was planned to assess kap and adherence of treatment of patients with primary hypothyroidism who have been diagnosed and receiving treatment for at least 3 months and visited our center for the first time . an observational cross - sectional study was conducted in the outpatient department of endocrinology clinic at maharaja agrasen hospital , punjabi bagh , new delhi , a 400-bedded teaching , superspecialty , national accreditation board for hospitals and healthcare providers and joint commission international accredited hospital . consecutive subjects of primary hypothyroid with age 18 years , who were on treatment for at least 3 months and visited our center first time , were invited to participate in the study . newly diagnosed patients , hypothyroidism secondary to pituitary / hypothalamic diseases , radioiodine ablation , surgery , neck irradiation , and drugs were excluded . pediatric patients < 18 years of age and patients with transient hypothyroidism were also excluded . patients with significant mental retardation , who were otherwise incapable of answering questionnaires , were also excluded from the study . a structured questionnaire was used to collect information on demographic parameters and to check subjects ' kap with respect to hypothyroidism and adherence of treatment . the questionnaire covered demographic parameters of study participants including their education level , duration of hypothyroidism , baseline tsh at diagnosis ; current dose of levo - t4 , current level of tsh if available , self - reported other comorbid medical conditions . the questionnaire covered issues such as meaning of medical terms thyroid , and hypothyroidism , patient 's knowledge about the basis of the treatment , monitoring during treatment , correct method of medication intake and belief in alternative medications , common beliefs and misconceptions about hypothyroidism , and common symptoms perceived by patients related to hypothyroidism . adherence to t4 therapy was assessed by asking the patients the number of doses missed in the last 1 month and was categorized as follows : adherent to treatment : missed < 5% dose in the last 1 monthmoderately adherent to treatment : missed 5% but < 15% dose in the last 1 monthnonadherent to treatment : missed 15% dose in the last 1 month . adherent to treatment : missed < 5% dose in the last 1 month moderately adherent to treatment : missed 5% but < 15% dose in the last 1 month nonadherent to treatment : missed 15% dose in the last 1 month . categorical variables were presented in number and percentage ( % ) , and continuous variables were presented as mean standard deviation . the data were entered into ms excel spreadsheet , and analysis was done using statistical package for social sciences ( spss ) ( chicago , illinois , usa ) version 21.0 software . categorical variables were presented in number and percentage ( % ) , and continuous variables were presented as mean standard deviation . the data were entered into ms excel spreadsheet , and analysis was done using statistical package for social sciences ( spss ) ( chicago , illinois , usa ) version 21.0 software . majority of participants belonged to the age group between 41 and 50 years ( 30.4% ) . median baseline tsh at diagnosis was 13.10 miu / l ( range : 320.8 ) . common associated comorbidities in these respondents were diabetes mellitus ( 24.8% ) and hypertension ( 23.6% ) . majority of participants were well educated , with 53.6% being graduates / postgraduates [ table 2 ] . demographic parameter of study population education level of study participants ( n=250 ) regarding knowledge of terminologies , out of 250 , only 88 ( 35.2% ) participants were aware of correct meaning of the term thyroid [ table 3 ] . similarly , only 128 ( 51.2% ) participants were aware about correct meaning of the term hypothyroidism . hypothyroidism as a swelling in the neck or increased hormone production by the thyroid gland . only 64 ( 25.6% ) participants knew correctly that t4 is used to replace and normalize the blood level of thyroid hormone . knowledge of terminologies related to thyroid of the study participants symptoms perceived related to hypothyroidism by study participants were weight gain ( 93.6% ) , easy fatigability ( 80% ) , irregular menstrual cycle ( 65.2% ) , infertility ( 55.6% ) , constipation ( 51.2% ) , excessive hair fall ( 41.2% ) , and skin problems ( 38% ) . nearly 11.6% participants believed that it can result in weight gain > 15 kg while another 18.8% believed that maximum weight gain can be between 10 and 15 kg . beliefs of study participants related to disease , its treatment and dietary precautions in the context of hypothyroidism are summarized in table 4 . regarding treatment , 20.4% participants believed that alternative therapy can cure hypothyroidism ; while 20.8% believed that iodized salt can be used to treat hypothyroidism . a large number of participants ( 40.8% ) believed that cabbage , cauliflower , and soya products should be avoided by patients with hypothyroidism . 9.6% participants felt that t4 can be stopped once lab reports return to normal level while 18.8% participants did not have any idea for same . regarding conception , 7.6% participants thought that hypothyroid women can not conceive , 62.8% thought they can conceive , while remaining 29.6% were not sure about it . a fair number of participants ( 22.8% ) did not follow the correct method of t4 administration with respect to meal ; 20.4% participants were not keeping minimum 30 min gap between t4 and meal ; while 2.4% were taking t4 either with meals or after meals . only 62.2% participants answered tsh as single best test for monitoring of treatment of primary hypothyroidism . regarding adherence , 90.4% participants were adherent to t4 , 2.4% were moderately adherent , and remaining 7.2% participants were nonadherent to t4 . this observational cross - sectional study was conducted to assess the kap and adherence to treatment in patients with primary hypothyroidism from a tertiary care teaching hospital in new delhi . knowledge and awareness about the disease among the patients is very important for better long outcome for any chronic disease . our study shows that a large number of patients with primary hypothyroidism lack basic knowledge about the disease ; fairly large number of patients have dietary as well as treatment - related false beliefs . kannan et al . did a pilot study of 34 hypothyroid patients from chennai , south india , to assess the kap among these patients . did kap study of 200 patients with thyroid swelling who attended cytology clinic at their institution . perumal et al . reported one cross - sectional study of 100 hypothyroid patients to assess their existing hypothyroidism related knowledge , health - seeking behavior , and health literacy among these patients . rai et al . reported one community - based cross - sectional study of 250 females of age group 1850 years from different areas of indore city of madhya pradesh belonging to different socioeconomic status and occupation to assess their knowledge about thyroid disorders , in this study only 20% females had either personal or family history of thyroid disorders . in our study , regarding the meaning of medical term thyroid , only 35.2% participants knew correctly that thyroid is a normal gland in the body , 29.6% considered thyroid as a hormonal disease , 9.2% as a swelling in the neck , and the remaining 26% did not have any idea about it . hypothyroidism is a clinical term for reduced secretion by thyroid gland , 11% participants thought hypothyroidism as a swelling in the neck , and 18% regarded it as increased secretion by thyroid gland , and remaining 31% had no idea regarding the same . in kannan study , 79.41% and 55.88% participants knew correct meaning of the terms thyroid and hypothyroidism , respectively . knowledge of disease - related basic terminologies is very important for patients to acquire further knowledge regarding their disease . in our study , only 25.6% participants knew correctly that t4 is used to replace and normalize the blood level of thyroid hormone while 36% participants considered it as means to stimulate thyroid gland to work normally . others considered that t4 was a drug to reduce neck swelling or for weight reduction . , 50% patients were aware that t4 is given to replace and normalize blood level of thyroid hormone . regarding treatment in our study , 9.6% participants felt that thyroid medication can be stopped once laboratory reports return to normal while 71.6% thought otherwise and the remaining 18.8% had no idea regarding the same . similarly , 8.4% patients felt that thyroid medication should be stopped during pregnancy as it can harm the fetus , 36.4% did not feel so and remaining 53.4% had no idea about same . , 32.35% patients felt that thyroid medication should be stopped once laboratory reports return to normal and 26.47% patients believed that thyroid medications should be stopped during pregnancy . among alternative medication use , 20.4% participants believed that alternative medicines such as ayurveda , yoga , unani , siddha , and homeopathy can cure hypothyroidism . our results are comparable to the previous studies , 20.58% and 35% participants with hypothyroidism had faith in alternative medications in studies by kannan et al . and singh et al . , respectively . among other misconceptions , 20.8% patients believed that hypothyroidism can be treated using iodized salt corresponding figures were much higher in a study by kannan et al . inappropriate knowledge regarding treatment leads to poor outcome of chronic diseases such as hypothyroidism . in india , considerable time needs to be spent in explaining the patient about the lifelong nature of the illness , need for long - term medication , and regular follow - up . in our study , 62.8% participants believed that hypothyroid female patients can conceive while 7.6% believed that hypothyroid women can not conceive and rest 29.6% were not sure about it . hypothyroidism is a common cause of infertility which can be easily managed by correcting the hypothyroid state . inadequate knowledge about pregnancy and hypothyroidism has created a certain amount of blemish in the minds of patients and their relatives , which leads to mental trauma as well as social differences in relationships , especially among young females . this needs to be addressed not only just by patient education at the level of physician but also at a social level in the form of awareness campaigns involving mass education . symptoms perceived related to hypothyroidism by participants were weight gain ( 93.6% ) , easy fatigability ( 80% ) , irregular menstrual cycle ( 65.2% ) , infertility ( 55.6% ) , constipation ( 51.2% ) , excessive hair fall ( 41.2% ) , skin problems ( 38% ) , and sore throat / neck pain ( 35% ) . patients commonly tend to attribute any symptom pertaining to neck ( commonly sore throat ) to their thyroid gland . the various misconceptions held by patients with respect to weight gain attributable to hypothyroidism are of clinical importance . it is well known that primary hypothyroidism does not cause an increase in weight gain of more than 35 kg . however , in our study , 11.6% participants believed that it can result in weight gain 15 kg while another 18.8% believed that weight gain can be between 11 and 15 kg . a study by kannan et al . and singh et al . also showed that a significant number of subjects believed that hypothyroidism causes excessive weight gain and obesity . however , in the previous studies , patient 's perception about quantitative weight gain in hypothyroidism was not assessed . physicians should always clarify to patients that hypothyroidism generally causes mild weight gain ( < 5 kg ) and should lay emphasis on the importance of diet and exercise for weight loss . among dietary misconceptions , 40.8% believed in dietary restrictions such as avoiding cabbage , cauliflower , and soya products . study results were almost similar to our study where 38.23% subjects believed in dietary restriction of cabbage , cauliflower , and soya products . in our study , 97.6% participants used to take t4 empty stomach in the morning while remaining 2.4% were taking it either with or after meal . almost 20.4% participants were not keeping minimum advised gap of 30 min between t4 ingestion and meals . regarding adherence , 90.4% participants were adherent to t4 , 2.4% were moderately adherent , and remaining 7.2% participants were nonadherent to t4 . it is well known that thyroid disease is not a major cause of mortality . however , hypothyroidism can contribute to morbidity from osteoporosis , hypercholesterolemia , cardiovascularm , and neuropsychiatric diseases . better knowledge and awareness regarding the disease in primary hypothyroid patients can significantly improve compliance of treatment and decrease the associated morbidity . in addition , this can further help even make patients spread correct facts and information regarding the disease to their relatives and friends in the society . there are a lot of factors contributing to lack of knowledge among patients in our country . lack of qualified physicians , less time spent by doctors on patient education because of high patient burden , lack of awareness , and not using available electronic media are some of them . lot of awareness is still required at physician level in developing countries like india for optimum management of hypothyroid patients . in addition , there is urgent need for endocrine nurses / counselors or dieticians who understand thyroidology and can spend time with patients explaining about their disease and its treatment . our study showed significant caveats in knowledge and awareness about the disease and its treatment in patients with primary hypothyroidism . in addition , a large number of patients had dietary as well as treatment - related misconceptions . public health measures are required to improve knowledge and awareness regarding the disease in patients with primary hypothyroidism .

the recently published paper by sanchez - sanchez and colleagues is a very interesting and well documented study showing how challenging it is to prevent fluid creep during early resuscitation after major burn trauma . under- and over - resuscitation have been associated with complications and poor outcome not only in patients with burns but in other critically ill patients as well . volume creep in major burns contributes to worsening of burn edema , conversion of superficial to deep burns , and compartment syndromes . although the use of vital signs heart rate and mean arterial pressure ( map ) and urine output may lead to under - resuscitation in burned patients who are critically ill , several teams have attempted to optimize fluid resuscitation after major burns [ 5 - 7 ] . basing fluid delivery on invasive hemodynamic monitoring they showed that fluid administration guided by a hemodynamics - oriented approach throughout the first 24-hour period limited to a urine output of 0.5 to 1 ml / kg per hour and a cardiac index of at least 2.2 l / minute per m was safe and resulted in less organ dysfunction . this year , snchez - snchez and colleagues present interesting results of a 3-year prospective cohort study of 132 consecutive critically ill patients ( age of 48 18 years ) with a burned body surface ( total body surface area , or tbsa ) of 35 22% . the resuscitation algorithm was guided by blood pressure ( map of more than 65 mm hg ) , urinary output ( 0.5 to 1 ml / kg ) , transpulmonary thermodilution , and lactate levels : the crude 4 ml / kg per tbsa formula was used as a starting value at the beginning of the resuscitation but was constantly adjusted to achieve ( a ) a cardiac index of more than 2.5 l / minute per m , ( b ) an intrathoracic blood volume index ( itbvi ) of more than 600 ml / m , and ( c ) normalization of lactate levels by 32 hours . the mean fluid rate required to achieve the protocol targets in the first 8 hours was 4.05 ml / kg per tbsa burned , increasing during the next 16 hours . the authors conclude that the initial burn trauma - induced hypovolemia is not optimally reflected by map and hourly urine output but can be detected by transpulmonary thermodilution during the early resuscitation phase . the authors consider that , thanks to their protocol , unnecessary fluid overload was prevented . however , there are some concerns with the results presented by snchez - snchez and colleagues and doubts about the achievement of what they presented as dry resuscitation , especially when strong endpoints such as intra - abdominal pressure and sepsis - related organ failure assessment ( sofa ) are considered . first , given a mean tbsa of 35% , the mortality rate is elevated ( 23% ) . second , 12% of patients have an abdominal compartment syndrome ( intra - abdominal pressure of more than 20 mm hg ) , of whom 50% die . third , acute renal failure occurred in 31.1% of patients and 11.4% finally required renal replacement therapy . fourth , the length of mechanical ventilation is high ( 21.5 days ) , and 24.2% of patients developed acute respiratory distress syndrome , despite a very low prevalence ( 9.1% ) of proven inhalation . the persistence of relatively high sofa scores on days 5 and 7 differs from the fast organ failure decay observed by arlati and colleagues with a stricter hypovolemia protocol . finally , early progressive enteral nutrition ( en ) that is , started within 12 hours of injury has been shown to improve outcome after major burns and should be integrated in any resuscitation protocol since it enhances gut perfusion in both animals and humans , reduces both pyloric dysfunction and gut edema , and hence improves gut function : further early en is associated with a significant reduction of the requirement for parenteral nutrition ( pn ) . in the spanish cohort , en was introduced only whenever possible , forcing the use of pn in 61.3% of patients , which is unusual and is likely to reflect gut dysfunction . despite showing the willingness to reduce fluid delivery indeed , the itbvi - driven resuscitation protocol results in a fluid delivery above the parkland - determined target , which by no means can be qualified as a hypovolemic target . remember that the advanced trauma life support ( atls ) guidelines now recommend considering the low range of 2 to 4 ml / kg per% tbsa of the parkland formula for calculation of the first 24 hours fluid administration ! furthermore , the resuscitation algorithm does mention the use of inotropes and vasopressors , but only after attempting changes in fluid load . this may be the missing link to explain higher - than - expected fluid administration . unfortunately , the results do not give details on the use of the latter or track the gain of weight . in conclusion , though not achieving the degree of over - resuscitation described by holm and colleagues , the strategy by snchez - snchez and colleagues still suffers from some degree of fluid overload and did not achieve the expected reduction of organ failures ( pulmonary , renal , and digestive ) . some degree of hypovolemia during the first 24 hours after burn does not require complete correction when occurring under close supervision . the atls proposed guidance by the low range of the parkland formula remains a good alternative to an invasive hemodynamic target to guide initial resuscitation after major burns . atls : advanced trauma life support ; en : enteral nutrition ; itbvi : intrathoracic blood volume index ; map : mean arterial pressure ; pn : parenteral nutrition ; sofa : sepsis - related organ failure assessment ; tbsa : total body surface area .

c - reactive protein ( crp ) , the prototypic acute phase reactant , is produced primarily by the liver as part of the body 's mechanism to restrict injury and promote repair after an inflammation evoking injury [ 13 ] . crp is a member of the phylogenetically ancient and evolutionarily conserved pentraxin family of proteins and consists of five noncovalently bound subunits , each of 206 amino acids , arranged symmetrically around a central pore . the molecule has a ligand recognition face that contains a ca - dependent binding site , and an effector molecule binding face that is capable of initiating fluid phase pathways of host defence ( by activating the complement system ) and cell - mediated ones ( by activating complement or binding to fc receptors ) . regulation of crp expression occurs mostly at the transcriptional level , with interleukin 6 ( il-6 ) being its major inducer and interleukin 1 ( il-1 ) synergistically enhancing the il-6 effect [ 4 , 5 ] . the rise in blood crp after tissue injury is rapid , with levels increasing by as much as 1000-fold above baseline within 24 hours . this plasticity makes blood crp an ideal clinical marker of a patient 's general health status , a purpose for which it has been used for half a century [ 16 ] . since the early 1980s , largely because of increasingly widespread use of automated high sensitivity crp assays , clinicians and physician scientists have been able to reproducibly and accurately measure the low levels of blood crp ( 3 mg this capacity has led to accumulation of extensive observational data linking crp to various kinds of disease [ 610 ] . the relationship of crp to the inflammatory aspects of cardiovascular disease ( cvd ) has been an area of keen interest . indeed , based upon multiple prospective epidemiological studies , crp is now recognized as an independent marker and powerful predictor for future risks of myocardial infarction ( mi ) , stroke , and death from coronary heart disease ( chd ) in individuals apparently free of known cvd . further , data from at least four clinical trials ( prove it - timi , reversal , jupiter , and saturn ) suggest a role for crp in the atherogenic process [ 1114 ] . in these studies , the indirect reduction of blood crp levels that accompanied treatment with statins was found to be independently and significantly related to event - free survival and/or decreased progression of documented coronary disease and/or major cardiovascular events . in patients where low density lipoprotein cholesterol ( ldl - c ) alone was reduced , disease progression was slowed by statin therapy , but in patients where ldl - c and crp were both reduced , atheroma progression was halted . furthermore in at - risk patients given maximally intensive statin therapy , lowering of crp was associated with atheroma regression . based on the known biology of crp it would not be a surprise if the protein was ultimately found to contribute to the pathophysiological processes leading to cvd . for example various studies demonstrate that crp can activate complement and endothelial cells and promote their dysfunction [ 1517 ] . others show that crp is detected in early atherosclerotic lesions [ 17 , 18 ] and that it is colocalized with activated complement components and enzymatically degraded ldl in human vascular lesions isolated by atherectomy [ 1921 ] . in addition there is compelling direct evidence from multiple transgenic models indicating that human crp has a pathogenic role in vascular disease [ 2225 ] . despite these data , generated independently by many different groups , the exact biological role of crp in cvd in humans and the overall importance of its contribution therein remains equivocal because there is no way to selectively reduce crp in patients . towards solving this nagging problem a small molecule inhibitor of human crp , 1,6-bis(phosphocholine)-hexane , was synthesized and tested in a preclinical rodent model in vivo . the compound is designed to crosslink two crp molecules , thereby increasing the protein 's clearance and blocking its ability to bind endogenous ligands . it was shown that in rats that were administered human crp by injection , administration of this compound ameliorated human crp associated exacerbation of mi caused by ligation of the coronary artery . despite the fact this was a xenogenic system , the study demonstrated for the first time that therapeutic inhibition of crp might be a promising new approach for cardioprotection in acute mi . still , to definitively address the question of causality of crp in the pathogenesis of cvd and whether reduction of crp would result in a meaningful decrease in adverse cvd outcomes , the use of a specific pharmacological inhibitor of endogenously expressed crp is preferred . antisense oligonucleotides ( asos ) are highly specific agents that can be used therapeutically to prevent the translation of disease - associated proteins , an effect achieved via selective degradation of targeted mrnas . because of their specificity and propensity to distribute to the liver , where crp is synthesized and secreted [ 35 ] , asos provide an efficient means of reducing crp expression . using crp transgenic mice ( crptg ) [ 29 , 30 ] we previously established that human crp targeting asos are effective for reduction of human crp and efficacious against collagen - induced arthritis . herein we employed additional species - specific crp targeting asos and tested their efficacy in two different animal models of cvd wherein a role for crp has already been established , namely , rats subjected to experimentally induced mi and crptg mice subjected to carotid artery ligation [ 24 , 25 ] . we provide new evidence that aso - mediated reduction of endogenously expressed rat crp ( in rats ) and human crp ( in crptg ) is efficacious in both diseases . crp specific asos have the potential to be therapeutically beneficial in humans at risk for cvd . eleven - week - old sprague - dawley rats were obtained from charles river breeding laboratories , fed a standard rat pellet diet ad libitum , and acclimated to local conditions for 1 week before use in any experiments . wild - type mice ( c57bl/6 strain ) and littermate crptg were from our own colonies . details of the human crp transgene and its human - like expression in crptg have been described elsewhere [ 29 , 30 ] . in crptg human crp is present in the blood at concentrations relevant to human disease , that is , low levels under steady - state conditions ( <3 g / ml ) and high levels during an acute phase response ( > 500 g / ml ) . all mice were fed a standard mouse pellet diet ad libitum and they were 812 weeks old when used in experiments . only male rats and male mice were subjected to experimentation and all were maintained at constant humidity ( 60 5% ) and temperature ( 24 1c ) with a 12 hour light cycle ( 6 am to 6 pm ) . all protocols were approved by the institutional animal care and use committee ( iacuc ) at the university of alabama at birmingham and were consistent with the guide for the care and use of laboratory animals published by the national institutes of health public health service policy on humane care and use of animals , dhew publication number 96 - 01 , phs policy revised in 2002 . asos designed to specifically hybridize to either rat or human crp mrna were synthesized and purified as described previously [ 28 , 31 , 32 ] . each aso was 20 nucleotides in length and comprised a central unmodified core consisting of 10 or 14 nucleotides , flanked by phosphorothioate linkages and three or five 2-o - methoxyethyl ( 2-moe ) modifications on the 3 and 5 flanking ends . the asos thus had a 3 - 14 - 3 or a 5 - 10 - 5 configuration . candidate asos were evaluated for their ability to reduce il-6 stimulated crp mrna expression in cultures of rat or human hepatoma cells ( data not shown ) . in these experiments several asos significantly reduced crp mrna levels , with ic50 values for the lead compounds in the 5 nm range ( data not shown ) . based on their potency in these assays , a rat crp specific aso ( isis 197178 ) and a human crp specific aso ( isis 353512 ) were chosen for evaluation in vivo . a third aso ( isis 141923 ) , which is a scrambled aso not complementary to any known rat or human gene sequence , served as a control . after acclimatization , rats were randomly assigned to 3 treatment groups and received for 4 wks the rat crp - specific aso isis 197178 or the control aso isis 141923 ( both at 150 mg / kg / wk i.p . ) or vehicle ( 0.9% nacl ) . at the beginning and end of the 4-week treatment phase blood was collected to assess crp , il-6 , and alanine transaminase ( alt ) levels . the next day echocardiography was performed and then the rats were subjected to left anterior descending coronary artery ( ladca ) ligation as described . briefly , rats were anesthetized with ketamine - xylazine ( 8015 mg / kg i.p . ) , intubated , ventilated with a rodent respirator , and laid on a heating pad warmed to 37c . the heart was exposed via a left intercostal thoracotomy through the 4th intercostal space , and the pericardium removed for identification of the ladca . the ladca was ligated 2 mm below the left atrium using a tapered needle and a 5 - 0 polypropylene ligature . occlusion was confirmed by a sudden pallor of the anterior wall of the left ventricle ( lv ) . the chest cavity was closed and the rats allowed to recover , with analgesic ( 0.05 mg / kg buprenorphine s.c . ) given twice daily over the next 3 days . one week after ladca echocardiography was repeated and the rats euthanized with an overdose of ketamine - xylazine . hearts were then removed , weighed , and cut into 2 mm slices ( average of 5 transverse slices / heart at the level below the ladca ) perpendicular to the apex - base axis . tissue slices were fixed with 4% paraformaldehyde , embedded in paraffin , cut into 5 m sections , and stained with picrosirius red ( 0.1% ) for assessment of collagen area ( an index of replacement fibrosis and infarct size ) . morphometric analysis of tissue sections was carried out by light microscopy with a qimaging qicam digital camera ( qimaging ) interfaced with a computer system running metamorph 6.2v4 software ( universal imaging ) . for infarct size estimation , the ratio of the picrosirius red - stained area ( or the perimeter of that area ) divided by the total area of the ventricle ( or the perimeter of the ventricle ) was calculated for six 5 m sections taken from 02 , 24 , 46 , 68 , and 810 mm distal from the heart apex . echocardiography was performed on isoflurane anesthetized rats using a philips sonos 5500 ultrasound system equipped with a 15 mhz transducer as described previously . after recording heart rate ( hr ) , lv end - systolic dimension ( lvesd ) and lv end - diastolic dimension ( lvedd ) were measured by two - dimensional - guided m - mode imaging from the parasternal short - axis view below the mitral valve . lv end - diastolic volume ( edv ) , end - systolic volume ( esv ) , ejection fraction ( ef ) , and fractional shortening ( fs ) were calculated as follows : edv = 7 lvedd/(2.4 + lvedd ) , esv = 7 lvesd/(2.4 + lvesd ) , fs = ( lvedd lvesd)/lvedd 100 , and ef = ( edv esv)/edv 100 . the mean velocity of circumferential shortening ( vcfr ) was also calculated . a single examiner blinded to treatment performed and interpreted all studies . wild - type versus crptg mice were randomly assigned to treatment groups to receive for 2 wks the human crp - specific aso 353512 ( 10 , 25 , or 50 mg / kg i.p . ) , the control aso isis 141923 ( 20 mg / kg / wk i.p . ) , or an equal volume vehicle ( 0.9% nacl ) . at the beginning and end of the 2 wk treatment phase blood was collected to assess human crp and il-6 . following the run - in phase the right common carotid artery ( rcca ) was ligated to stimulate neointima generation as described previously [ 24 , 25 ] . briefly , the rcca was exposed through a midline cervical incision and ligated with an 8 - 0 silk suture just proximal to the bifurcation . the left common carotid artery was surgically exposed but not ligated and served as an internal control . treatments continued postsurgically for 2 more weeks and then the mice were anesthetized and euthanized with an overdose of pentobarbital . the vasculature was immediately flushed with 0.01 m sodium phosphate buffer ( ph 7.4 ) and perfused with 10% formalin and both carotid arteries were excised , fixed in 10% formalin , embedded in paraffin , and sectioned . representative serial sections were stained with hematoxylin and eosin ( h&e ) and examined under a light microscope to locate the ligature site ; then additional sections of the artery taken 200 , 350 , 500 , and 700 m proximal to the ligation site were identified and treated with verhoeff 's elastin stain to enhance the elastic laminae . sections of the unligated contralateral vessels were obtained and processed in the same fashion ( data not shown ) . computer - assisted morphometric analysis of digitized images captured from each arterial section was performed with image analysis software ( scion image ) . to calculate vessel patency , the cross - sectional area of the vessel lumen was divided by the cross - sectional area of the area bounded by the internal elastic lamina . all measurements were performed by a single examiner blinded to the genotype and treatment of the mice . rat crp was measured using a commercially available enzyme - linked immunoassay kit and the manufacturer 's instructions ( emd millipore , darmstadt , germany ) and human crp was measured by an elisa as described previously [ 24 , 25 ] . alt was determined using infinity alt reagents ( sigma - aldrich corp . , st . pairwise comparisons were done using student 's t - tests , and comparisons among multiple experimental groups were performed with one - way anova followed by pairwise multiple comparisons using the protected least - squares difference test . all statistical analyses were performed using the sigmastat software package ( sigmastat , jandel scientific ) . blood crp level in rats ( n = 36 ) was 321.9 18.9 g / ml at baseline , with no significant difference among the three randomly assigned treatment groups ( anova ) . administration of the rat crp specific drug isis 197178 ( n = 19 rats ) reduced blood crp by an average 65% - from 303.8 29.5 g / ml at baseline to 90.4 17.2 g / ml on day 28 ( figure 1(a ) ) . in comparison , rats treated with saline ( n = 8) or the control aso isis 141923 ( n = 9 ) showed no significant deviation of blood crp from baseline levels on day 28 ( figure 1(a ) ) . indeed during this time period crp levels increased , rather than decreased , in rats receiving saline ( 10% increase from baseline ) and isis 141923 ( 24% increase ) . il-6 level at baseline and on day 28 were highly variable with no significant differences among the three treatment groups ( anovas ) ( figure 1(b ) ) . importantly , there was no reduction of il-6 ( the major inducer of rat crp ) in rats treated with isis 197178 ( figure 1(b ) ) . both the control and the crp - specific asos were well tolerated as judged by absence of elevation of blood alt ( figure 1(c ) ) . ladca ligation on day 28 caused blood crp to increase by 33% by day 35 for saline - treated rats and by 13% for isis 141923-treated rats , but the elevation of crp was completely blocked for rats receiving isis 197178 ( figure 1(a ) ) . after eliminating rats that died during surgery or within hours after ladca ligation due to lethal mi ( 3/19 isis 197178 treated rats , 3/9 isis 141923 treated rats , and 2/8 saline treated rats ) and after inspecting post - mi echocardiograms and stained heart sections to eliminate rats in which the ladca was not properly ligated so no mi was achieved ( 4/16 isis 197178 treated rats , 1/6 isis 141923 treated rats , and 2/6 saline treated rats ) , 21 rats remained for further study . for statistical purposes the remaining saline - treated and isis 141923-treated animals ( n = 9 in total ) were pooled and compared to the remaining isis 197178-treated rats ( n = 12 ) . echocardiographic indices in this subset were consistent with a subtle but significant improvement in post - mi cardiac function for rats treated with isis 197178 , which showed a 1423% improvement in ef , fs , and vcfr ( figure 2 ) . consistent with the echocardiography findings , infarct size was reduced in rats treated with isis 197178 compared to controls ( figure 3 ) , with a small but significant zone of protection observed proximal to the site of ladca ligation ( i.e. , 610 mm from the apex of the heart ) ( figure 3 ) . these results show that for rats treated with isis 197178 , an aso that targets rat crp mrna and thereby lowers circulating crp protein level effectively without causing toxicity or inflammation , cardiac dysfunction resulting from experimentally induced acute mi was reduced . in these experiments crptg mice were treated for 2 weeks with different doses of a human crp specific aso ( isis 353512 ; doses of 10 , 25 , or 50 mg / kg / week i.p . ) versus a control aso ( isis 141923 at 20 mg / kg / week i.p . ) or vehicle ( 0.9% saline ) . following this 2-week run - in phase therapy continued for 2 more weeks and the ligated vessels were harvested to quantitate the effect of human crp lowering on the blood vessel injury response . serum human crp level in crptg mice ( n = 69 ) was 12.7 1.03 g / ml at baseline with no statistically significant difference ( anova ) among the various treatment groups . for both saline - treated and isis 141923-treated crptg mice , serum human crp level increased above baseline by ~30% by day 14 and by ~4085% by day 28 ( two weeks after rcca ligation ) ( figure 4 ) . in contrast for mice receiving isis 353512 , the increase in human crp level was prevented at the 10 mk / kg / wk dose and reversed in a dose - dependent fashion at the higher doses ( figure 4 ) . like the aso we tested in rats , the human crp specific aso isis 353512 neither reduced serum il-6 nor raised serum alt levels in crptg mice ( data not shown ) . furthermore , isis 353512 did not reduce mouse crp levels ( data not shown ) . for crptg mice subjected to surgery , patency of the ligated rcca was significantly improved by treatment with the human crp lowering aso isis 353512 ( figure 5(a ) ) . in stark contrast isis 353512 had no effect on ligated rcca patency in mice that did not express human crp ( figure 5(b ) ) . these results show that for crptg mice treated with isis 353512 , an aso that targets human crp mrna and thereby lowers circulating human crp protein level effectively without causing toxicity or inflammation , carotid artery stenosis resulting from blood vessel ligation is reduced . the association of elevated baseline crp to increased risk of chd is widely recognized [ 3537 ] , but it is not known if this association is causal . furthermore if the association is causal it is not known which of the many biological actions of crp might support the effect . notwithstanding the many informative studies of crp biology performed in vitro and in animal models , a clinically approved and specific inhibitor of human crp will be needed before a true understanding of the physiologic role of crp in humans at risk of chd can be ascertained . if treatment of at - risk patients with a crp lowering drug is found to lower the risk , then that would certainly settle the ongoing debate about whether crp plays a role in cardiovascular disease . on the other hand even if lowering crp has no protective effect , the predictive association would still remain . a small molecule inhibitor of crp [ 1,6-bis(phosphocholine)-hexane ] that occludes the ligand - binding b face of crp and thereby reportedly blocks its ability to activate complement was tested preclinically . since ( i ) complement activation is known to occur in concert with cardiovascular disease [ 20 , 21 ] , ( ii ) complement activation products are found in vascular lesions associated with cardiovascular disease , and ( iii ) crp is known to activate complement and colocalize with complement fragments deposited in vascular lesions , then administration of 1,6-bis(phosphocholine)-hexane should be of benefit in cvd . indeed in rats , this compound was shown to prevent the exacerbating effect of exogenously administered human crp on experimentally induced mi . however , since crp also interacts with fcrs , treatment with 1,6-bis(phosphocholine)-hexane may not completely block all of the potentially detrimental functions of the protein . also it is not clear what consequences circulating crp decamers ( two pentamers crosslinked by 1,6-bis(phosphocholine)-hexane ) might have on the vasculature , since presumably these complexes could be deposited and potentially cause autoimmune or inflammatory side effects . therefore , despite the fact that this drug improved infarct size and improved cardiac function in rats receiving human crp , these shortcomings could ultimately limit its therapeutic potential in man . rather than depleting the circulating protein from the blood , the asos we used inhibit crp production by specifically and selectively preventing the translation of crp mrnas . the aso approach has been successfully used to target proteins not readily amenable to small molecule or antibody based therapeutic interventions . for example kynamro , an antisense inhibitor of apolipoprotein b , the principal apoprotein present on all atherogenic lipids , was approved by the fda in january 2013 for use as an adjunct to first - line lipid - lowering therapies in homozygous familial hypercholesterolemia [ 3941 ] . because asos have much longer half - lives compared to small molecule inhibitors [ 41 , 42 ] furthermore , crp is well suited for inhibition using aso technology because the protein is synthesized primarily by hepatocytes [ 3 , 4 , 29 , 34 ] , cells that readily accumulate antisense drugs and are sensitive to aso pharmacology [ 4244 ] . asos also accumulate in extrahepatic cells and tissues known to make crp , such as the kidney , alveolar macrophages , and adipocytes , [ 19 , 37 , 42 ] . the asos we tested here were designed to target rat crp and human crp ( in crptg mice ) . each aso was well tolerated at all doses and in both species tested , and both crp targeting inhibitors specifically reduced their respective crp serum levels after only short - term administration with a modest dosing regimen . we did not test complement levels in the current study , but we have shown previously that aso mediated lowering of human crp is accompanied by reduced complement activation in crptg mice , an effect like that of 1,6-bis(phosphocholine)-hexane in rats . notably , aso - mediated reduction of crp in both species was not due to reduction of il-6 and each of the tested asos blunted ( or reversed ) the rise in serum crp caused by surgery . the proven ability of antisense inhibitors to reduce baseline expression of rat crp in rats and human crp in crptg mice and to block crp upregulation after surgery , after treatment for a short duration with only low doses of antisense drugs , suggests that these agents should be useful for intervention in both chronic and acute disease processes . indeed , we have previously shown that another human crp specific aso ( isis 329993 ) is efficacious in a crptg mouse model of inflammatory arthritis . we have shown here for the first time that , in both rats and mice , aso - mediated lowering of crp results in improved outcomes following cardiovascular insult . despite a body of evidence that crp level is a fairly strong predictor of cvd , there is still no agreement on how to integrate crp measurement into clinical practice or indeed whether it should even routinely be evaluated [ 4648 ] . the main reason is that it is still unknown whether crp plays a pathophysiologic role in cvd in humans . without a human specific - crp drug it has not been possible to conduct clinical trials to test the crp - cvd hypothesis . the crp - specific aso inhibitors that we describe here could fill this gap and provide the impetus for future in vivo pharmacological , toxicological , and ultimately clinical studies that will help clearly delineate the role of crp in cvd in humans . if future studies do confirm a role for crp in cvd , then perhaps a reasonable aso treatment group would be patients whose admission values of crp are 10 mg / l or greater , as these are the patients at highest risk for death and mi .